Systematic identification, classification, and characterization of the open reading frames which encode novel helicase-related proteins inSaccharomyces cerevisiae by gene disruption

Yeast 15, 219–253 (1999)

Systematic Identification, Classification, andCharacterization of the Open Reading Frames whichEncode Novel Helicase-related Proteins inSaccharomyces cerevisiae by Gene Disruption andNorthern AnalysisAKIKO SHIRATORI1,2, TAKEHIKO SHIBATA2,3, MIKIO ARISAWA4, FUMIO HANAOKA1,5,YASUFUMI MURAKAMI1 AND TOSHIHIKO EKI1*1Cellular Physiology Laboratory, Tsukuba Life Science Center, The Institute of Physical and Chemical Research(RIKEN), 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan2Graduate School of Science and Engineering, Saitama University, 255 Shimo-okubo, Urawa, Saitama 338-0825,Japan3Cellular and Molecular Biology Laboratory, The Institute of Physical and Chemical Research (RIKEN),2-1 Hirosawa, Wako, Saitama 351-0106, Japan4Department of Mycology, Nippon Roche Research Center, 200 Kajiwara, Kamakura, Kanagawa 247-0063, Japan5Institute for Molecular and Cellular Biology, Osaka University, Suita, Osaka 565-0871, Japan

Helicase-related proteins play important roles in various cellular processes incuding DNA replication, DNA repair,RNA processing and so on. It has been well known that the amino acid sequences of these proteins contain severalconserved motifs, and that the open reading frames (ORFs) which encode helicase-related proteins make up severalgene families. In this study, we have identified 134 ORFs that encode helicase-like proteins in the Saccharomycesgenome, based on similarity with the ORFs of authentic helicase and helicase-related proteins. Multiple alignmentof the ORF sequences resulted in the 134 ORFs being classified to 11 clusters. Seven out of 21 previouslyuncharacterized ORFs (YDL031w, YDL070w, YDL084w, YGL150c, YKL078w, YLR276c, and YMR128w) wereidentified by systematic gene disruption, to be essential for vegetative growth. Three (YDR332w, YGL064c, andYOL095c) out of the remaining 14 dispensable ORFs exhibited the slow-growth phenotype at 30)C and 37)C.Furthermore, the expression profiles of transcripts from 43 ORFs were examined under seven different growthconditions by Northern analysis and reverse transcription-polymerase chain reaction, indicating that all of the 43tested ORFs were transcribed. Interestingly, we found that the level of transcript from 34 helicase-like genes wasmarkedly increased by heat shock. This suggests that helicase-like genes may be involved in the biosynthesis ofnucleic acids and proteins, and that the genes can be transcriptionally activated by heat shock to compensate for therepressed synthesis of mRNA and protein. Copyright ? 1999 John Wiley & Sons, Ltd.

— Saccharomyces cerevisiae; ATPase; expression profile; essential gene; functional genomics; genefamily; helicase; gene disruption; heat shock; phylogenetic analysis; slow growth phenotype; systematic analysis;transcriptional activation; YBR245c; YDL031w; YDL070w; YDL084w; YDR291w; YDR332w; YDR334w;YER176w; YFR038w; YGL064c; YGL150c; YHR031c; YIR002c; YKL017c; YKL078w; YLR247c; YLR276c;YLR419w; YMR128w; YNL218w; YOL095c; YOR304w

*Correspondence to: T. Eki, Cellular Physiology Laboratory,Tsukuba Life Science Center, The Institute of Physicaland Chemical Research (RIKEN), 3-1-1 Koyadai, Tsukuba,Ibaraki 305-0074, Japan. Tel.: +81-298-36-9059; fax: +81-298-36-9137; e-mail: [email protected]

CCC 0749–503X/99/030219–35 $17.50Copyright ? 1999 John Wiley & Sons, Ltd.

INTRODUCTION

The budding yeast Saccharomyces cerevisiae isan important model organism for studying the

Received 9 April 1998Accepted 14 August 1998

220 . .

basic biological processes of higher eukaryotes.Although the yeast is a simple eukaryote and itsgenome size is relatively small, the molecularmechanisms of yeast, such as control of cell cycle,signal transduction, DNA replication, tran-scription and DNA repair, are similar to those ofhigher eukaryotes. World-wide yeast genomesequencing efforts have led to the discovery ofapproximately 6000 genes in yeast (Goffeau et al.,1997; Mewes et al., 1997), and this allows us toidentify and classify the ORF sequences into genefamilies by function or sequence motif (Henikoffet al., 1997). ORFs that are classified intoappropriate gene families, such as ABC proteins(Decottignies and Goffeau, 1997) or proteinkinases (Hunter and Plowman, 1997), are suitablefor further functional analysis, because thesimilarity in sequence and conserved motifs toknown ORFs can be used to deduce the biologicalfunction of newly identified ORFs.

In this study, we applied this approach to ORFswhich encode DNA/RNA helicase-like proteins.A number of studies have indicated that DNA/RNA helicases and helicase-related proteins areinvolved in multiple cellular processes such asDNA replication, DNA repair, DNA recombi-nation, transcription, pre-mRNA splicing, rRNAsynthesis, translation, chromatin remodeling andso on. For instance, Ssl2p, Rad3p and Hpr5p areDNA helicases involving in DNA repair, Pif1pacts as a DNA helicase for mitochondrial DNArepair, and Dna2p helicase is involved in DNAreplication. Both Tif1p and Tif2p are RNA heli-cases which are essential for translation, Nam7pacts as an RNA helicase for mRNA decay, andPrp22p is an essential RNA helicase for pre-mRNA splicing (see Table 1). It has also been wellknown that many helicase-like proteins cooper-atively act in numerous sequential steps of thereactions as the multiprotein complexes, such asthe spliceosome, TFIIH, chromatin remodelingcomplexes, and so on.

Helicases commonly share Walker A and Bdomains with the ATPase superfamily. The con-served Walker domains are essential for NTP-binding of NTP-hydrolysing enzymes. Sequenceanalysis of a number of DNA/RNA helicases andhelicase-like proteins have led to the identificationof helicase subfamilies, each subfamily havingseveral conserved motifs (i.e. helicase motifs I–VI),such as the DEAD-box protein family (Schmidand Linder, 1992; Gorbalenya and Koonin, 1993).A role of each specific domain has been clarifying

Copyright ? 1999 John Wiley & Sons, Ltd.

by a number of biochemical analyses using mutantproteins and X-ray crystallographic studies ofthree DNA helicases (Marians, 1997).

Since DNA/RNA helicases play important rolesin every cellular process that involves the manipu-lation of nucleic acids, from a functional point ofview, helicase-like proteins are essential for life andhave been highly conserved throughout evolution.These essential roles, in which helicases andhelicase-like proteins are involved, have beenclarified throughout genetic studies using manytemperature-sensitive (ts) mutants which displaydefects in cell growth, like prp mutants (Rymondand Rosbach, 1992). Furthermore, recent progressin the genome project has led to the discovery ofa number of helicase-like proteins in manyorganisms, although the function of many proteinsare, as yet, unknown. This indicates that helicase-related proteins have expanded and evolved, anddiverged into protein families that have variouscellular functions (Gorbalenya and Koonin, 1993).Especially, many helicase-like proteins seem toplay an important role in the transcriptional regu-lation of proteins that are involved in developmentand cell differentiation in higher eukaryotes. Forinstance, the Drosophila maleless protein is anRNA helicase essential for dosage compen-sation of the X chromosome (Lee et al., 1997).The human ARTX protein, whose amino acidsequence contains SNF2-like helicase motifs,suggests that the ARTX protein is involved in thetranscriptional regulation of the genes, includingthe á-globin gene (Gibbons et al., 1995).

In addition, the study of helicase-like proteins inyeast is important for human medical researchbecause of the conservation of their structuresand functions among species. It has been reportedthat genetic dysfunction of a human helicase(or putative helicase) protein, leads to inheritedhuman disorders (Ellis, 1997). For example, theamino acid sequences of the human WRN andBLM proteins contain the DEAH-box; these pro-teins are E. coli RecQ-like DNA helicases (Suzukiet al., 1997; Karow et al., 1997); Werner’s syn-drome is associated with genetic dysfunction of theWRN protein, which leads to an accelerated age-ing phenotype (Yu et al., 1996); Bloom’s syndromeis associated with genetic dysfunction of the BLMprotein, which leads to a hypermutation pheno-type (Ellis et al., 1995). The closest homologue inyeast of both human genes is SGS1, which encodesa DNA helicase (Lu et al., 1996a); deletion ofthe SGS1 gene in yeast also results in increased

Yeast 15, 219–253 (1999)

221 -

chromosome instability and premature ageing(Sinclair and Guarente, 1997; Sinclair et al., 1997).

Thus, it is important to identify all of the geneswhich encode helicase-like proteins in yeast and tocharacterize newly discovered genes to find theircellular function. Here, we have identified andclassified more than 134 ORFs which encode yeasthelicase-like proteins. For newly identified ORFs,the phenotype of the null mutant of each ORF wasexamined, as well as the pattern of expressionof each ORF under a variety of physiologicalconditions. We have tried to deduce the func-tion of newly identified ORFs throughout theseanalyses.

MATERIALS AND METHODS

Screening of helicase-related open reading frames(ORFs)

Open reading frames (ORFs) that belong to thefollowing three categories were defined as ‘thehelicase-like ORFs’: Category #1, ORFs whichencode the well-characterized DNA and RNAhelicases or nucleic acid-dependent ATPases;Category #2, ORFs which encode proteins thatbelong to known helicase families, previouslydescribed by Gorbalenya and Koonin (1993) (i.e.proteins containing typical motifs, such as motifsI, Ia–VI, which are widely conserved in helicases).The ORFs encode the replication factor C (RFC)proteins (Cullmann et al., 1995), minichromosomemaintenance (MCM) proteins (Koonin, 1993a),and the Sug1p, which encodes an ATPase subunitof the 26S proteasome (Weeda et al., 1997), belongto this group, because they contain the devianthelicase-motifs in their sequences; Category #3, anORF which exhibits meaningful homology insequence to an authentic ORF of Category #1 or#2. The ORFs of Categories #1 and #2 wereidentified by performing keyword searches in thepublic Saccharomyces cerevisiae genome database,Saccharomyces Genome Database (SGD; Cherryet al., 1998), the Yeast Protein Database (YPD;Hodges et al., 1998) and the MIPS database(Mewes et al., 1998). A homology search usingeach of the ORFs in Categories #1 and #2 wascarried out using the BLASTP program (Altschulet al., 1990) of WU-BLAST version 2, against theamino acid sequences of translated yeast ORFs inthe SGD (http://genome-www2.stanford.edu/cgi-bin/SGD/nph-blast2sgd). An ORF whoseputative amino acid sequence has a BLAST


probability (p) value of power than 1·0#e"5, wasdefined as a helicase-like ORF and placed inCategory #3.

Sequence analysisAll of the helicase-like ORFs were aligned using

the sequence alignment program, CLUSTAL Wversion 1.7 (Thompson et al., 1994). The ORFsclassified into 11 clusters (I–XI) based on similarityin the protein sequence coded by the ORF. TheTREEVIEW program (version 1.5) was used todraw the phylogenetic tree (Page, 1996). Multiplealignment of the sequences in Cluster V was car-ried out by using the PILEUP program; the resultswere drawn by the PRETTYBOX program (GCGpackage, version 8.1).

The putative localization of a protein within theyeast cell was predicted by the PSORT program(Nakai and Kanehisa, 1992), which is availablethrough the internet (http://cookie.imcb.osaka-u.ac.jp/nakai/psort.html). The TMpred program(Hofmann and Stoffel, 1993) was used to deter-mine if a particular protein has transmembranesegments. A search was performed using thepolypeptides expressed by the newly identifiedhelicase-like ORFs in the PROSITE database, todetermine its putative features (Bairoch, 1993); theconserved regions and functional domains of theseproteins were identified by performing a BEAUTYsearch (Worley et al., 1995), in the client-serversystem of the Human Genome Center at theBaylor College of Medicine. Other manipulationsof the ORF sequences were carried out by usingthe GENETYX MAC program package (version9; Software Development Co., Tokyo).

StrainsThe diploid strain RAY3A-D (MATa/MATá

ura3/ura3 leu2/leu2 his3/his3 trp1/trp1) was used forthe gene disruption experiment, as well as forNorthern analysis of the ORFs under the sporula-tion condition. Strain RAY3A-D was constructedfrom R27-7-C-D (MATa/MATá ura3/ura3 leu2/leu2 trp1/trp1), and RAY1-13D (MATa ura3 leu2trp1 his3) (Tanaka et al., 1988). Haploid strainsW303-1a (MATa ade2-1 ura3-1 his3-11 trp1-1leu2-3, 112 can1) and NOY396á (MATá ade2-1ura3-1 his3-11 trp1-1 leu2-3, 112 can1), were usedfor disrupting non-essential genes (both of thesestrains were kindly provided as gifts from Dr H.Uemura, NIBH, MITI). The haploid strain S288C(MATa mal gal2) was used for Northern analysis

Yeast 15, 219–253 (1999)

222 . .

of the ORFs under all of the conditions except forthe sporulation condition. The haploid strainsLP2915-9B (MATa cdc9-2 ade2-1 his3-D200leu2-3, 2-112 trp1-D ura3-52) and 185-3-4g (MATacdc28-1 leu2) were used as ts control strains forphenotype analysis; they were purchased fromthe Yeast Genetic Stock Center (University ofCalifornia at Berkeley, Berkeley, CA).

Disruption of ORFs and tetrad analysisDisruption of yeast ORFs was carried out by

using a PCR-based gene replacement approach(Figure 1), where the Candida glabrata HIS3 genewas used as the selection marker (Kitada et al.,


1995). This method is suitable for efficient genedisruption because there is low probability thatunexpected integration into the chromosomalHIS3 locus would occur. For each ORF, a pair ofolignucleotides (forward and reverse primers) wasconstructed. The forward primer (60 mer) con-tained the two 40-nucleotide sequences on eitherside of the first ATG codon of the ORF, and a3*-tail of 20 nucleotides (5*-CACCGATCAACGTACAGTGG-3*) corresponding to the 5*-sequenceof the Candida glabrata HIS3 gene (Kitada et al.,1995). The reverse primer contained the unique40-nucleotide sequence near the stop codon of theORF and a 3*-tail of 20 nucleotides (5*-TGACAA

Figure 1. Outline of the gene disruption procedure. The entire region ofthe targeted ORF was deleted, and replaced by C. glabrata HIS3, by thePCR-mediated gene replacement approach. An outline of the steps isillustrated. PCR using a pair of chimeric oligonucleotides (60 bp each)was performed to prepare the DNA fragment containing the selectablemarker C. glabrata HIS3, used for transforming yeast diploid RAY3A-D.Two procedures were performed to verify the accurate replacement of theORF into the transformants: (1) PCR using two sets of primers, i.e. OFand C.g His-R; OR and C.g His-F; and (2) Southern analysis using theORF-specific probes.

Yeast 15, 219–253 (1999)

223 -

TCTGGCAGCTCGCT-3*) corresponding to the3*-sequence of the HIS3. PCR was carried outusing each pair of primers (10 ì), and 5 ng ofcloned C. glabrata HIS3 DNA as the template inthe reaction (0·4 ml) in the presence of ExTaqpolymerase (12·5 units, TaKaRa) for 40 cycles(denaturation: 94)C, 30 s; annealing: 50)C, 30 s;elongation: 72)C, 30 s). Approximately 1 ìg of thePCR product was used to transform lithiumacetate-treated yeast cells, according to the methodof Gietz et al. (1995), without the carrier DNA.The transformed cells were spread on agar platescontaining CM (complete minimal) drop-outmedium without histidine, and kept for 2–3 days at30)C.

The His+ transformants were tested by PCRand Southern analysis to verify correct replace-ment in the targeted locus. In the PCR reaction,oligonucleotides OF and OR were generated fromthe 5* and 3* sites, respectively, that flank theparticular ORF. Oligonucleotides C.g His-F (5*-ATATGCTAGTGGTGATGGGCA-3*) and C.gHis-R (5*-GATGTGCAAGTCCCCTATACA-3*),were generated from the sequence of C. glabrataHIS3. Colony PCR using the OF and C.g His-Rprimers, or the OR and C.g His-F primers, gener-ates a product of known size in the presence ofgenomic DNA (Figure 1). Colony PCR using theOF and C.g His-R primers was performed underthe reaction conditions noted above, and usingExTaq polymerase (0·25 units). PCR with the ORand C.g His-F primers was carried out in a reac-tion mixture that also contained ExTaq polym-erase (2·5 units), and TaqStart antibody (0·55 ìg,Clontech), for 38 cycles (annealing 94)C, 30 s;elongation 68)C, 1 min, per cycle).

For Southern analysis, genomic DNA was puri-fied from the transformant cells, and from twocontrol strains, RAY3A-D and S288C. 5 ìg ofHindIII-digested genomic DNA was separated by0·7% agarose gel electrophoresis, and then trans-ferred to a Hybond-N+ membrane (Amersham).A blotted filter was hybridized with 32P-labelledDNA covering the tested ORF, or the C. glabrataHIS3 fragment (633 bp in size) of PCR using theC.g His-F2 primer (5*-ATGGCGTTTGTTAAGAGGGTTACG-3*) and C.g His-R2 primers (5*-CTATGCTAGGACACCCTTAGTGG-3*) as theprobe. The ORF-specific probe was prepared bylong PCR with a set of ORF-specific primers.Restriction fragments with the expected sizes fromboth the wild-type and correctly replaced alleles,were detected in genomic DNA samples from all of


the transformants established from RAY3A-D.The size of each fragment was consistent with therestriction map of nucleotide sequences in theSGD database. The nucleotide sequences ofPCR primers for gene disruptions and hybridiz-ation probes are available from the author uponrequest.

In tetrad analysis, two independent disrupt-ant clones for each ORF were cultured in 1%potassium acetate for 4–5 days at 30)C, to germi-nate spores, dissected under the microscope, andthen were incubated on YPD plates, at 30)C for2–3 days, to test whether they were viable.

Disruption of dispensable gene and phenotypicanalysis

To observe the influence of a null mutation ofa non-essential gene, we generated a series ofdisruptants from two haploid strains, W303-1aand NOY396á, with the same background exceptfor a mating-type. Disruption of each dispensableORF, and subsequent verification of genotype,were performed as described above. The cells werecultured in a YPD medium containing 0·004%adenine sulphate (i.e. YPAD). At least two nullmutant lines which were indepenently isolated,were incubated on the YPAD plate at 30)C and at37)C for 2 days, to test whether any growth delayhad occurred.

Preparation of total RNA

We prepared three types of culture media inwhich intact yeast cells were cultured for extract-ing RNA (Kaiser et al., 1994): (1) SC (syntheticcomplete) medium (Naitou et al., 1997); (2) SD(synthetic dextrose minimum medium); and (3)minimal sporulation medium (1% potassiumacetate). The yeast cells were cultured under theseven different conditions described below, prior toRNA extraction. Total RNA was extracted andpurified by the methods described by Naitou et al.(1997).- Condition 1: Vegatative growth. Yeast strain

S288C was cultured at 30)C in SC medium. Thecells were harvested when they were in theexponential growing phase (OD600=0·8–1·0).

- Condition 2: Nutrient starvation at low tempera-ture. Strain S288C was cultured at 16)C in SDmedium. The cells were harvested when theywere in the exponential growing phase.

Yeast 15, 219–253 (1999)

224 . .

- Condition 3: Heat shock. Strain S288C wascultured at 30)C in SC medium in the expo-nential growing phase. The cells were incubatedat 39)C for 10 min in a water bath and then wereharvested.

- Condition 4: Sporulation. Yeast diploid strainRAY3A-D was cultured at 30)C in SC medium.The cells were collected by centrifugation whenthey were in the exponential growing phase. Thecells were resuspended in minimal sporulationmedium (1% potassium acetate) and cultured at25)C with slow shaking for 2 days prior toharvest.

- Condition 5: Ultraviolet (UV) irradiation. Expo-nentially growing S288C cells in SC medium at30)C were collected by centrifugation andwashed with distilled water. Approximately3#1010 cells were suspended in 8 ml of fresh SCmedium and spread on to SC plates. Afterirradiation with UV light (254 nm, 120 J/m2)using the UV Stratalinker 1800 (Stratagene), thecells were collected from the plates, washed withwater and then cultured in fresh SC medium at30)C for 1·5 h.

- Condition 6: Methylmethane sulphonate (MMS)treatment. S288C cells were cultured in SCmedium at 30)C. When the cells were in theexponential growing phase, they were treatedwith 0·01% (w/v) MMS (Sigma) for 2 h and thenwere harvested.

- Condition 7: Hydroxyurea (HU) treatment.S288C cells were cultured in SC medium at30)C. When the cells were in the exponentialgrowing phase, they were treated with 50 mHU (Sigma) for 2 h prior to harvest. These cellswere compared with cells that did not receiveMMS or HU treatment.

Northern analysisNorthern analyses were performed using

RNA samples (30 ìg) from yeast cells which hadbeen cultured in the above seven conditions, asdescribed by Naitou et al. (1997). All of the DNAprobes used for Northern analysis were preparedby PCR using primers that were specific to eachORF. (Note: Information of primer sequences andPCR conditions are available from the authorupon request). The lengths of the DNA probeswere adjusted to approximately 500 bp. The radio-activity of all of the DNA probes that wereused for the hybridization was adjusted to a similarlevel (0·5–1·0#107 cpm). The radioactivity of the


hybridized signal was quantified by using the Bio-imaging system BAS2000 (Fuji Film Co.), thedetailed method of which is described elsewhere(Naitou et al., 1997). In short, the radioactivity ofthe hybridized signal of each condition on thesingle blot was adjusted by adding a knownamount of rRNA, which itself was quantified bydensitography. The relative amount of transcriptwas determined by comparing it with the amountof the control transcript of YFL007w, or that ofYFL039c (ACT1), on the same membrane. Thelength of the mRNA of YFL007w, at 5·4 kb, islarge enough to separate most of the tested tran-scripts; furthermore, this transcript is expressed inall of the seven conditions tested. The relativequantity of transcript in the seven growth con-ditions were then determined, and compared withthe quantity of transcript in the control condition.In this study, we examined the expression pro-files of the 19 newly-identified ORFs exceptfor YMR128w and YNL218w, plus the follow-ing seven ORFs whose functions are unknown(as of March 1998) under the above con-ditions: YDL160c [DHH1], YER176w [ECM32],YGR271w, YHR169w [DBP8], YKR024c [DBP7],YMR290c [HAS1] and YNR038w [DBP6]. Thefollowing control conditions were used for each ofthe seven conditions used for Northern analysis:the amount of transcript in the vegetative growthcondition (Condition 1 in ‘Preparation of totalRNA’) was used as the control for determining thelevel of transcript in the conditions of nutrientstarvation, heat shock and sporulation. Formeasuring the quantity of transcript in the UVirradiation and drug treatment conditions, thequantity of transcript in the condition withouttreatment, was used as the control. To evaluate thequality of the RNA preparation in each condition,the transcripts of the following ORFs wereemployed as a control: (1) YFL039c (ACT1) formeasuring the quantity of transcript; (2) YFL016cfor the heat-shock induced transcript (Naitouet al., 1997); (3) YJL026w (RNR2) and YIL066c(RNR3) for the UV-induced and the drug-inducedtranscripts (Elledge et al., 1993); (4) YFR032c forsporulation-specific transcript; (5) YFL022w(FRS2), YFR050c (PRE4) and YFR052w (NIN1)for use as controls to compare with our previousdata (Naitou et al., 1997).

Transcripts from the following 17 yeast ORFswith known biological function, were also charac-terized in our study by Northern analysis, in thesame seven growth conditions: DNA repair

Yeast 15, 219–253 (1999)

225 -

(YBR114w [RAD16], YER171w [RAD3],YGL163c [RAD54], YIL143c [SSL2]); DNA repli-cation (YGL201c [MCM6]; transcriptional regula-tion (YER164w [CHD1]); chromosomesegregation (YBR073w [RDH54]); mitochondrialfunction (YML061c [PIF1]); pre-mRNA splicing(YBR237w [PRP5], YER013w [PRP22], YGL120c[PRP43], YNR011c [PRP2]); mRNA transport(YJL050w [MTR4]); and rRNA processing(YDR021w [FAL1], YGL078c [DBP3], YGL171w[ROK1], YJL033w [HCA4]). Finally, the expres-sion profiles of 43 helicase-like ORFs wereobtained by systematic Northern analysis.

Reverse transcription-polymerase chain reaction(RT-PCR)

The transcripts from the four ORFs(YMR128w, YJL092w, YJR035w and YLR032w),which could not be detected by Northern analysis,were detected by RT-PCR. RT-PCR was per-formed using mRNA purified from vegetatively-growing S288C cells and a pair of ORF-specificprimers (the primers used for the preparation ofDNA probes for Northern analysis) as describedby Naitou et al. (1997).

RESULTS

Identification of the helicase-like ORFs inSaccharomyces cerevisiae

A survey in the publicly available Saccharo-myces genome databases (SGD, YPD and MIPS)was performed to search for ORFs which encode:(1) DNA/RNA helicases or nucleic acid-dependentATPases (14 known helicases and 24 nucleic acid-dependent ATPases in Table 1); (2) proteins con-taining helicase-related sequence motifs such as theDEAD or DExH boxes, which are involved innucleic acid metabolism (Gorbalenya and Koonin,1993); and (3) proteins which have signifi-cant similarity to one of the authentic helicasesequences. The search for helicase-related proteinsresulted in 134 ORFs (i.e. 2·1% of total ORFs inyeast genome) (see Table 1).

Out of the 134 ORFs, 21 ORFs exhibit signifi-cant similarity in sequence; these ORFs are com-monly encoded in the Y* sequence of the telomere.These ORFs are specifically transcribed in themeiotic phase; however, their functions in yeastcells are unknown and some of these Y*-encodedhelicase-like ORFs are pseudogene (Louis, 1995).The following 20 ORFs, out of the remaining 113


unique ORFs (17·7% of the ORFs), are newlyidentified ORFs which have not yet beengenetically or biochemically characterized (as ofMarch 1998): YBR245c, YDL031w, YDL070w,YDL084w, YDR291w, YDR332w, YDR334w,YFR038w, YGL064c, YGL150c, YHR031c,YIR002c, YLL034c, YLR247c, YLR276c,YLR419w, YNL218w, YOL095c, YOR304w andYPL074w. Although helicase-like proteins havebeen genetically and biochemically characterizedby a number of laboratories, the cellular functionof the proteins encoded by 38 ORFs (33·6% of the113 unique ORFs), which includes these 20 ORFs,remain to be determined.

Questionable ORFsThrough searching the databases for helicase-

like proteins, we found a few ORFs that arecategorized as being ‘helicase’, although whethereach of these proteins actually has helicase activity,is questionable. For instance, Lu et al. (1996b)reported DNA helicase activity associated withHmo1p, the protein encoded by YDR174w. How-ever, Hmo1p is probably a homologue of theHMG1/2 proteins in higher eukaryotes based onits amino acid sequence, and the recombinantHmo1p was inactive as a helicase (Lu et al.,1996b). Pro3p has also been reported to havehelicase activity (YPD); however, it is unlikely thatPro3p is actually a helicase, because Pro3p is adelta 1-pyrroline-5-carboxylate reductase whichconverts pyrroline-5-carboxylate to proline in theproline biosynthesis pathway (Brandriss andFalvey, 1992). Ssu72p has also been reported tohave similarity with an ATP-dependent RNA heli-case (Sun and Hampsey, 1996); however, theBLAST p-value of the protein encoded by SSU72against all known yeast protein sequences, wasvery high (the smallest BLAST p-value was 0·25,to YOL148c). Therefore, these ORFs were omittedin this study.

Phylogenetic classification of helicase-relatedORFs

The 134 yeast helicase-like ORFs were multiplyaligned using the computer program CLUSTALW. The resultant phylogenetic tree is shown inFigure 2. Based on similarity in amino acidsequence, the 134 ORFs were grouped into 11clusters (I–XI). The 11 clusters themselves weregrouped into three large clusters (I–V, VI–VIII andIX–XI). Within each cluster, several subclusters of

Yeast 15, 219–253 (1999)

226 . .

Table 1. Helicase-like ORFs in Saccharomyces cerevisiae.

SubclusteraORFname

Genenameb Chr.

ProteinDB

MW(kDa)c

ATPase activityhelicase activity (polarity) Complex and interactionsd

I.1 YBR245c ISW1 II P38144 132·7YER164w CHD1 V P32657 168·2YIL126w STH1 IX P32597 156·7 RSC complex; Rsc8p, Sfh1p (P)YOR290c SNF2 XV P22082 194·0 DNA-dep ATPase SWI/SNF complex; Swi3p, Snf11p, Anc1p, Rsc8p (P),

Gal11p, Rgr1p, Spt7p, Spt20p, Swi4p (G)YOR304w ISW2 XV P38144 130·3

I.2 YBR073w RDH54 II P38086 108·0 Dmclp (P)YGL163c RAD54 VII P32863 101·8 DNA-dep ATPase Rad51p (P), Hpr5p, Pol3p, Pol30p (G)YJR035w RAD26 X P40352 124·5 DNA-dep ATPase

I.3 YAL019w FUN30 I P31380 128·5YDR334w IV S70099 174·5YFR038w VI P43610 88·7YGL150c INO80 VII P53115 171·4

I.4 YPL082c MOT1 XVI P32333 209·9 TBP-stimulated ATPase TBP-DNA complex (P); Spt15p (P),Toa1p, Spt3p, Spt6p, Spt7p (G)

I.5 YBR114w RAD16 II P31244 91·4 Rad7p, Smd3p, Msl1p (P)YLR032w RAD5 XII P32849 134·0 ssDNA-dep ATPase Soh1p (P)YOR191w RIS1 XY S67083 184·4 Sir4p, Dmc1p, Prp9p (P)

I.6 YGR252w GCN5 VII Q03330 51·1 SAGA complexAdr1p, Ada2p, Spt20p, Ngg1p, Ire1p, Hfi1p (P),Afr1p, Swi1p (G)

YIL143c SSL2 IX Q00578 95·3 DNA-dep ATPase,ATP-dep DNA helicase (3*]5*)

TFIIH component; Rad2p, Rad3p, Msl1p (P)

I.7 YHR031c VIII P38766 81·6YML061c PIF1 XIII P07271 92·9 ssDNA-dep ATPase,

ATP-dep DNA helicase (5*]3*)

II.1 YGR056w RSC1 VII P53236 106·7 RSC complex; Hsh49p, Yor100cp (P)YLR357w RSC2 XII S51465 102·3 RSC complex; Mud2p (P)

II.2 YDL070w BDF2 IV S67605 72·5 Bdf1p (G)YLR399c BDF1 XII P35817 77·0 Bdf2p (G)

II.3 YBR081c SPT7 II P35177 152·5 SAGA complexYKR008w RSC4 XI Q02206 72·3 RSC complex

III.1 YBL023c MCM2 II P29469 98·7 MCM complexYBR202w CDC47 II P38132 94·8 MCM complexYEL032w MCM3 V P24279 107·5 MCM complexYGL201c MCM6 VII P53091 112·9 MCM complex; Mcm10p (P)YLR274w CDC46 XII P29496 86·4 MCM complexYPR019w CDC54 XVI P30665 105·0 MCM complex

III.2 YMR190c SGS1 XIII P35187 163·8 ATP-dep DNA helicase Top2 (P), Top3p (P, G)

IV.1 YDL007c RPT2 IV P40327 48·8 ATPase ProteasomeYDR394w RPT3 IV P33298 47·9 ProteasomeYER017c AFG3 V P39925 82·2 mtAAA protease; Yta12p (P)YGL048c RPT6 (SUG1) VII Q01939 45·3 ATPase ProteasomeYKL145w RPT1 XI P33299 52·0 ProteasomeYMR089c YTA12 XIII P40341 88·4 mtAAA protease; Afg3p (P)YOR117w RPT5 XV P33297 48·2 ProteasomeYOR259c RPT4 XV S67156 49·4 ProteasomeYPR024w YME1 XVI P32795 81·8

IV.2 YGR270w YTA7 VII P40340 157·4IV.3 YBR080c SEC18 II P18759 84·1 á-SNAP; Sec17p (G, P), Pep12p (P)IV.4 YDL126c CDC48 IV P25694 91·9 Doa1p, Ufe1p (P)

YER047c SAP1 V P39955 100·3 Spt2p (P)YGR028w MSP1 VII P28737 40·2YKL197c PEX1 XI P24004 117·3YLL034c XII S64785 93·1YLR397c AFG2 XII P32794 84·7YNL329c PEX6 XIV P33760 115·6YPL074w YTA6 XVI P40324 85·3 Glc7p (P)YPR173c VPS4 XVI S59831 48·2 Vma2p (G)

IV.5 YDR375c BCS1 IV P32839 51·0

Copyright ? 1999 John Wiley & Sons, Ltd. Yeast 15, 219–253 (1999)

227 -

Table 1. Continued

Motifse FunctionsDisruptant phenotypesf

(March 1998) References

DEAH/ATP1/leu zipper NDDEAH/ATP1/chromodomain Transcriptional regulation Slightly slow growth (6-azauracil resistant) Woodage et al. (1997)DEGH/ATP1/bromodomain Transcription/remodeling factor Lethal Laurent et al. (1992)DEGH/ATP1/bromodomain Transcription,

chromatin remodelingViable Laurent et al. (1993)

DEAH/ATP/leu zipper NDDEGH/ATP1 Repair/recombination Viable (recombination defact in diploid) Klein (1997)DEGH/ATP1/leu zipper Repair/recombination Viable (X-ray sensitive) Petukhova et al. (1998)DEGH/ATP1 Repair (transcription coupled) Viable

(mutator, delayed recovery after UV damage)Guzder et al. (1996)

DEGH/ATP1 Repair Viable (UV resistant) Clark et al. (1992)DEAH/ATP1/leu zipper NDDEGH/ATP1 ND Eki et al. (1996)DEAQ/ATP ND James et al. (1995)DEGH/ATP1/leu zipper Transcriptional modulator Lethal Auble et al. (1997)

DEAH/ATP1/zinc finger (C3HC4) Excision repair Viable (UV sensitive) Schild et al. (1992)DEGH/ATP1/zinc finger (C3HC4) Repair (post replication) Viable (UV, X-ray sensitive) Johnson et al. (1994)DEGQ/ATP1/zinc finger (C3HC4) Silencing Viable (lower mating switching rate) Zhang and Buchman (1997)Bromodomain Transcriptional activation Viable Grant et al. (1997)

DEVH/ATP1 Transcription,transcription-coupled repair

Lethal Guzder et al. (1994)

DEIS/ATP NDDEIS/ATP mtDNA repair,

telomere maintenanceViable

(long telomere, mtDNA repair defect)Lahaye et al. (1991)

Chromatin remodeling Slow growthChromatin remodeling Slow growth

Bromodomain NDBromodomain Sporulation Slow growth (sporulation defect) Chua and Roeder (1995)Bromodomain/ATP2 Transcriptional activation Slow growth Grant et al. (1997)Bromodomain Chromatin remodeling Lethal

DEGH/ATP2 Replication (initiation) Lethal Chong et al. (1996)DEFD (MCM)/ATP2 Replication (initiation) Lethal ibid.DEFD (MCM)/ATP2 Replication (initiation) Lethal ibid.DEFD (MCM)/ATP2 Replication (initiation) Lethal ibid.DEFD (MCM)/ATP2 Replication (initiation) Lethal ibid.DEFD (MCM)/ATP2 Replication (initiation) Lethal ibid.DEAH/ATP Chromosome segregation

and maintenanceViable

(hyper-recombination, accelerated aging)Lu et al. (1996a);

Sinclair and Guarente (1997)

DEID/ATP Protein degradation Lethal Confalonieri and Duguet (1995)DEVD/ATP Protein degradation Lethal ibid.DEID/ATP Protein degradation Viable ibid.DEID/ATP Protein degradation Lethal ibid.DEID/ATP Protein degradation Lethal ibid.DEID/ATP Protein degradation Viable ibid.DELD/ATP Protein degradation Lethal ibid.DEVD/ATP Protein degradation Lethal ibid.DELD/ATP Viable ibid.DEID/ATP Viable ibid.DELD/ATP Secretory pathway Lethal ibid.DEID/ATP ER homotypic fusion Lethal ibid.DEID/ATP Viable ibid.DEID/DEFD/ATP Viable ibid.DEFD/ATP Peroxisome biogenesis ND ibid.DEID/ATP ND ibid.DEID/ATP/leu zipper Lethal ibid.ATP Peroxisome biogenesis ND ibid.DEID/ATP ND ibid.DEVD/ATP Endocytosis, membrane transport Viable ibid.ATP Viable ibid.


228 . .

Table 1. Continued.

SubclusteraORFname

Genenameb Chr.

ProteinDB

MW(kDa)c


V.1 YER176w ECM32 V P32644 127·0 ssDNA-dep ATPase,ATP-dep DNA helicase (5*]3*)

YKL017c HCS1 XI P34243 78·3 DNA-dep ATPase,ATP-dep DNA helicase (5*]3*)

DNA polymerase á (P)

YMR080c NAM7 XIII P30771 109·4 DNA/RNA-dep ATPase,DNA/RNA helicase

mRNA decay complex (Nmd2p and Upf3p),Dbp2p, Nmd1-5p, Sup35p, Sup45p, Upf3p, Snp1p (P)

V.2 YLR430w SEN1 XII Q00416 252·5 tRNA (rRNA) spliceosome component,Smd3p (P)

V.3 YHR164c DNA2 VIII P38859 171·7 DNA-dep ATPase,ATP-dep DNA helicase (3*]5*)

FEN1 nuclease (P), Rad27p (P, G),Spt16p, Tor1p, Pob1p (G)

VI.2 YDR291w IV S70120 123·5VI.3 YDR332w IV S59797 78·5

YIR002c IX P40562 114·0

VII.1 YDL031w DBP10 IV Q12389 113·2YDL084w SUB2 IV Q07478 50·3 Brr1p (G)YDL160c DHH1 IV P39517 57·5 Pop2p (P), Pkc1p (G)YDR021w FAL1 IV Q12099 45·2YFL002c SPB4 VI P25808 69·4 60S ribosomal subunit (P)YHR065c RRP3 VIII P38712 60·9 RNA-dep ATPase (weak)YHR169w DBP8 VIII P38719 47·9YJL033w HCA4 X P20448 87·2YJL138c TIF2 X P10081 44·7 RNA-dep ATPase,

ATP-dep RNA helicaseTranslation initiation factor,

Ded1p, Tif1p (G)YKR059w TIF1 XI P10081 44·7 RNA-dep ATPase,

ATP-dep RNA helicaseTranslation initiation factor,

Ded1p, Cdc33p, Tif2p, Tif3p (G)YLL008w DRS1 XII P32892 82·0 Orc2p (G)YMR290c HAS1 XIII Q03532 56·7 Set1p (P)YOR046c DBP5 XV P20449 53·9 RNA-dep ATPase,

ATP-dep RNA helicaseVII.2 YBR237w PRP5 II P21372 96·4 RNA-dep ATPase Spliceosome (P)

YDR243c PRP28 IV P23394 66·6 RNA-dep ATPase (?)g SpliceosomeYGL078c DBP3 VII P20447 58·8YGL171w ROK1 VII P45818 63·7 Snr10p, Gar1p (G)YNL112w DBP2 XIV P24783 61·0 Nam7p (P), Pol2p (G)YOR204w DED1 XV P06634 65·6 Srm1p (P), Tif1p, Tif2p, Cdc33p (G)YPL119c DBP1 XVI P24784 67·9 Ded1p (G)

VII.3 YBR142w MAK5 II P38112 87·0

VII.4 YLR276c DBP9 XII Q06218 68·1VII.5 VDR194c MSS116 IV P15424 76·3

YKR024c DBP7 XI P36120 83·3 Dbp6p (G)VII.6 YGL064c VII P53166 63·1

YNR038w DBP6 XIV P53734 70·4 Dbp7p (G)

VIII.1 YJL050w MTR4 X P47047 122·0 Rrp4p (G)YLR398c SK12 XII P35207 146·0 Dcp1p, Kem1p (G)

VIII.2 YPL029w SUV3 XVI P32580 81·4 mt Exoribonuclease complex

VIII.3 YER172c BRR2 V P32639 246·1 Spliceosome; Cus1p (P), Snp1p (G, P)YGR271w VII P53327 224·8

VIII.4 YGL251c HFM1 VII P51979 118·8

IX.1 YJR068w RFC2 X P40348 39·7 RFC complex; Pol30p (P)YNL290w RFC3 XIV P38629 38·2 ssDNA-dep ATPase RFC complex; Pol30p (P)YOL094c RFC4 XV P40339 36·1 RFC complex; Pol30p (P)

IX.2 YOR217w RFC1 XV P38630 94·9 RFCIX.3 YBR087w RFC5 II P38251 39·9 RFC complex; Pol30p (P)IX.4 YNL218w XIV P40151 66·5

X.1 YER013w PRP22 V P24384 130·0 DNA-dep ATPase,ATP-dep RNA helicase

Spliceosome; Msl5p (P)

YGL120c PRP43 VII P53131 87·5 SpliceosomeYKL078w JA2 XI P36009 82·7YKR086w PRP16 XI P15938 121·6 RNA-dep ATPase

ATP-dep RNA helicaseSpliceosome; Cdc40p, Ecm2p, Slu7p,

Prp8p, Prp18p (G)YMR128w ECM16 XIII Q04217 144·9YNR011c PRP2 XIV P20095 99·8 RNA-dep ATPase Spliceosome (P); Spp2p (P)


229 -

Table 1. Continued



DExxQ/ATP Slightly slow growth(cell wall, membrane defect)

Bean and Matson (1997);Lussier et al. (1997)

DExxQ/ATP Replication (?) ND Biswas et al. (1997)

DExxQ/ATP mRNA decay Viable (respiration defect) Jacobson and Peltz (1996)

DExxQ/ATP/leu zipper tRNA/rRNA processing Lethal Steinmetz and Brow (1996)

DExxQ/ATP Replication (elongation) Lethal Budd and Campbell (1995)

DELH/ATP NDDEAH/ATP1 NDDEAH/ATP1 ND

DEAD/ATP ND Saren et al. (1997)DECD/ATP NDDEAD/ATP Viable Strahl-Bolsinger and Tanner (1993)DEAD/ATP 18S rRNA processing Lethal Kressler et al. (1997)DEAD/ATP1 25S rRNA maturation Lethal Sachs and Davis (1990)DEAD/ATP 18S rRNA processing Lethal O’Day et al. (1996a)DEAD/ATP LethalDEAD/ATP 18S rRNA processing Lethal Liang et al. (1997)DEAD/ATP Translation Viable (lethal with TIF1 disruption) Schmid and Linder (1991)

DEAD/ATP Translation Viable (lethal with TIF2 disruption) ibid.

DEAD/ATP 25S rRNA maturation Lethal Ripmaster et al. (1992)DEAD/ATP LethalDEAD/ATP mRNA export Lethal Chang et al. (1990); Tseng et al. (1998)DEAD/ATP Pre-mRNA splicing Lethal O’Day et al. (1996b)DEAD/ATP/leu zipper Pre-mRNA splicing Lethal Strauss and Guthrie (1994)DEAD/ATP 25S rRNA maturation Slow growth Weaver et al. (1997)DEAD/ATP 18S rRNA processing Lethal Venema et al. (1997)DEAD/ATP Slow growth Barta and Iggo (1995)DEAD/ATP Translation Lethal Chang et al. (1997)DEAD/ATP Viable Jamieson and Beggs (1991)DEAD/ATP 60S ribosomal subunit biosynthesis

dsRNA killer plasmidLethal Wickner (1996)

DEVD/ATP NDDEAD/ATP mtRNA splicing Viable (respiration defect) Seraphin et al. (1989)DEGD/ATP 60S ribosomal subunit biosynthesis Slow growth Daugeron and Linder (1998)DEAD/ATP NDDEAD/ATP 60S ribosomal subunit biosynthesis Lethal Kressler et al. (1998)

DEVH/ATP mRNA transport Lethal Liang et al. (1996)DEVH/ATP Antiviral protein mRNA decay Lethal Wickner (1996);

Anderson and Parker (1998)DEIQ/ATP mtRNA processing Viable (mtDNA loss) Stepien et al. (1992)DEIH/ATP/leu zipper Pre-mRNA splicing Lethal Noble and Guthrie (1996)DEVH/ATP Viable Martegani et al. (1997)DEIH/ATP1 Viable Coissac et al. (1996)

DEAD/ATP Replication (elongation) Lethal Cullmann et al. (1995)DEAD/ATP Replication (elongation) Lethal Li and Burgers (1994)DEAD/ATP Replication (elongation) Lethal Cullmann et al. (1995)DEVD/ATP/leu zipper Replication (elongation) Lethal ibid.NEAN/ATPh Replication (elongation) Lethal ibid.DEID/ATP ND Coster et al. (1995)

DEAH/ATP Pre-mRNA splicing Lethal Schwer and Gross (1998)

DEAH/ATP Pre-mRNA splicing Lethal (ts) Arenas and Abelson (1997)DEAH/ATP ND Company et al. (1991)DEAH/ATP Pre-mRNA splicing Lethal Schwer and Guthrie (1991); Wang et al. (1998)

DEAH/ATP ND Lussier et al. (1997)DEAH/ATP Pre-mRNA splicing Lethal (ts) Kim et al. (1992)


230 . .

sequences were determined based on similarity insequence. Table 1 notes the motifs which arepresent in the protein encoded by each ORF, aswell as the function of each ORF (if known). Fourlarge clusters (I, IV, VI and VII) were detected inthe tree. Clusters I and VII contain many ORFsencoding SNF/RAD-like proteins and typicalDEAD-box proteins, respectively. The Y*-encodedORFs make up Cluster VI and most of the ORFsin Cluster IV, encode the Sug1p-related proteins.In addition, we found that the proteins in ClusterV conserve the deviant Walker-type NTP-bindingmotif B. Alignment of the amino acid sequences ofall five proteins encoded by the ORFs of Cluster V,


reveals that each of the five proteins contains atleast eight conserved domains (I, Ia–V, Va, andVI) of the ‘superfamily I helicases’ (Koonin, 1992;Koonin and Rudd, 1996) (see Figure 3). Basedon the amino acid sequence of the deviantNTP-binding motif B (i.e. the helicase motif II), wedefined this helicase subfamily as the ‘DExxQfamily’.

Systematic gene disruption of newly-identifiedORFs that encode helicase-like proteins

Since many helicase and helicase-like proteinsare involved in important cellular processes such

Table 1. Continued.

SubclusteraORFname

Genenameb Chr.

ProteinDB

MW(kDa)c


X.2 YLR419w XII S59384 163·1X.3 YMR078c CTF18 XIII P49956 84·3

XI.1 YJL092w HPR5 X P12954 134·3 DNA-dep ATPase,ATP-dep DNA helicase (3*]5*)

Msl1p (P), Rad54p (G)

YOL095c HM11 XV S57374 80·6XI.2 YLR247c XII S59393 180·3

XI.3 YER171w RAD3 V P06839 89·7 ssDNA-dep ATPase,ATP-dep DNA helicase (5*]3*)

TFIIH component, repairosome,Ssl1p, Ssl2p (P), Kin28p, Tfb3p (G)

YPL008w CHL1 XVI P22516 98·8

Y* subtelomeric ORF familyi

VI.1 YBL111c II S70303 76·2 Unknown UnknownYBL113c II S70305 87·0YDR545w IV S67814 203·8YEL077c V S70306 143·1YER190w V P40105 190·5YFL066c VI P43538 43·8YGR296w VII P53345 211·1YHL050c VIII P38721 79·0YHR218w VIII P38899 68·9YHR219w VIII P38900 70·1YIL177c IX P40434 197·5YJL225c X P40889 197·6YLL066c XII S50953 135·6YLL067c XII S64819 135·2YLR466w XII S70310 156·4YLR467w XII S65004 203·8YML133c XIII Q12054 31·6YNL339c XIV S63325 211·1O7535 XV P24088 203·8YPL283c XVI P53345 211·1YPR204w XVI S65341 115·1

Note: Selected ORFs exhibit the BLAST probability scores (smaller than 1·0#e"5) against the sequences of the authentic helicases and the helicase-relatedproteins (DEAD/DExH family, etc.) by BLAST search. mtDNA, mitochondrial DNA.aSubcluster in each cluster was determined by the results of multiple alignments as shown in Figure 2.bBDF2, DBP10, HM11, INO80, ISW1 and ISW2 were reported during paper submission (from April to July 1998).cCalculated molecular weight of protein.dReported protein–protein interactions and components of the multiprotein complex: MCM, minichromosome maintenance; RFC, replication factor C; RSC,remodel the structure of chromatin; SAGA, SPT-ADA-GCN5-acetyltransferase. (P), physical interactions (two-hybrid interaction, co-purification, etc.); (G),genetic interactions (synthetic lethality, high copy suppression, etc.).

Footnotes to Table continued on next page

Yeast 15, 219–253 (1999)

231 -

as DNA replication or translation, a null mutationin a gene which encodes a helicase-related proteinis often lethal to the organism. A null mutation inone of 47 ORFs, of the 88 ORFs which have beencharacterized to date, causes the lethal phenotype;null mutants of the remaining 41 genes are viableunder normal growth conditions (see Table 1). Atpresent (March 1998), phenotypes of the nullmutant of 25 out of the 113 unique ORFs (exclud-ing the 21 helicase-like ORFs encoded in the Y*region), have not been determined.

To study whether the newly identified helicase-related ORFs are essential for cell growth, weperformed a systematic disruption of these ORFs


(except for four SUG1-related ORFs) by using thePCR-mediated gene replacement procedure. TheHIS3 gene of Candida was used as a selectablemarker to prevent integration into the originalHIS3 locus. Disruptants from diploid strainRAY3A-D were cultured in a sporulation mediumto germinate spores; this was followed by tetradanalysis. The viability of the dissected spores weretested on a YPD plate at 30)C. The null mutationsof seven out of tested 21 ORFs, YDL031w,YDL070w, YDL084w, YGL150c, YKL078w(JA2), YLR276c (DBP9) and YMR128w(ECM16), led to lethal phenotypes. Deletants ofthe remaining 14 ORFs (YBR245c, YDR291w,

Table 1. Continued



DEVH/ATP NDDEID/ATP Chromosome segregation,

telomere maintenanceSlow growth

(cs, short telomere, hyper-recombination)Kouprina et al. (1994)

DEFQ/ATP/leu zipper Repair, recombination Viable(mutator, repair defect, sporulation defect)

Rong and Klein (1993)

DEFQ/ATP NDDEVQ/ATP1,

zinc finger (C3HC4)/leu zipperND

DEAH/ATP1 Transcription-coupled repair Lethal Sung et al. (1987)

DEAH/ATP1 Chromosome segregation Viable (abnormal chromosome segregation) Gerring et al. (1990)

DEFH/ATP Unknown ND

DEFH/ATPDEFH/ATPDEFH/ATPDEFH/ATPDEFH/ATPDELH/ATPDEFH/ATP

DETHDETHDEFH/ATPDEFH/ATPDEFH/ATPDEFH/ATPDEFH/ATPDEFH/ATPDEFH/ATPDEFH/ATPDEFH/ATP

Footnotes to Table continued from previous pageeAmino acid sequence motifs: ATP, Walker NTP-binding motif A ([A/G]xxxxGK[S/T], PS00017); ATP1, deviant motif A (GxGK[S/T]); ATP2, deviant motif A(GxxxxxK[S/T]) (Koonin, 1993b); DExD/H, ATP-dependent helicases signatures (Walker motif B, PS00690); DEFD (MCM), MCM family signature (PS00847;Koonin, 1993b); DExxx, putative Walker motif B; bromodomain (PS00633), chromodomain (PS00598), and C3HC4 type zinc finger (RING finger) signature(PS00518) are also indicated.fPhenotype of null mutants which have been characterized are briefly described. ND, not determined.g(?), indicated, but not confirmed.hDetermined by alignment data.iThese ORFs in Y* subtelomeric regions encode helicase-like proteins.

Yeast 15, 219–253 (1999)

232 . .

Copyri
ght ? 1999 John Wiley & Sons, Ltd. Yeast 15, 219–253 (1999)

233 -

YDR332w, YDR334w, YFR038w, YGL064c,YHR031c, YIR002c, YKL017c [HCS1],YLR247c, YLR419w, YNL218w, YOL095c and


YOR304w) were viable (data was presented forreview). The HIS3 markers co-segregated withtheir null phenotypes. Tetrad analysis results in

Figure 3. Conserved motifs among the five proteins which make up the ‘DExxQ family’ (Cluster V). The five proteinsequences (YER176w, YKL017c, YMR080c, YLR430w, and YHR164c) were aligned by the PILEUP program;alignment was drawn by the PRETTYBOX program. Identical amino acid residues among all five proteins areindicated by closed boxes; similar amino acid residues are indicated by shadow boxes. The numbers indicate theposition of the amino acid residue in the protein. Roman numerals I–VI correspond to the helicase-conserved motifsreported by Gorbalenya and Koonin (1993). Motif Va was reported by Koonin and Rudd (1996). The amino acidresidues of the Walker-type NTP-binding site A (Koonin, 1993b), and those of the putative Walker-type motif B(motif II), are indicated by the open triangles and open circles, respectively.

Figure 2. Phylogenetic tree of the helicase-related proteins in Saccharomyces cerevisiae. Through searching the databases, 134ORFs which encode helicase-like proteins were identified. These ORFs were multiply aligned using the CLUSTAL W program,and the phylogenetic tree was drawn by the TREEVIEW program. The resultant 11 clusters (I–XI) are indicated. The ORF andname of the gene (if the ORF has been previously named) are indicated. The ORFs were classified by biological function into 14groups; these functions are indicated by the colour boxes, each colour indicating a particular function (see ‘DISCUSSION’). Thescale of 0·1 indicates 0·1 nucleotide substitutions per site.

Yeast 15, 219–253 (1999)

234 . .

four colonies of disruptants. Following is the resultof tetrad analysis of each of the seven essentialORFs: two colonies of disruptants were viable on aYPD plate, but did not grow on the His" CMplate. The result from tetrad analysis of a non-essential ORF, was that two out of four viablecolonies were not able to grow on a selection plate(data not shown). Thus, we identified seven new,essential, helicase-like ORFs and 14 ORFs that aredispensable.

Helicase-like ORFs that are not essential for cellgrowth; the growth characteristics of yeast whichcontain null mutations of these ORFs

We identified 14 ORFs which are not essentialfor cell viability. To observe the phenotype of thenull mutation of each of the dispensable ORFs, wegenerated a series of disruptants of these ORFsfrom two haploid strains, W303-1a and NOY396á,of the same genetic background except for matingtype. The growth rate of the null mutant of each ofthese ORFs, was examined on YPAD plates at30)C and 37)C. Interestingly, the growth rate ofthe haploid W303-1a cells bearing each of the nullalleles of YDR332w, YGL064c and YOL095c, wasdecreased at 30)C, in comparison with the wild-type cells; at 37)C, the defect of growth rates ofthree null mutants of both the W303-1a cells andthe NOY396á cells, were much enhanced at 37)C.(Figure 4 shows the growth rate of the null mu-tants in the W303-1a cells.) We failed to find anydifference in growth characteristics between the nullmutant in the W303-1a strain and the same nullmutant in the NOY396á strain (data not shown).

Detection of transcripts from newly-identifiedORFs which encode helicase-like proteins, bysystematic Northern analysis and RT-PCR

Northern hybridization and RT-PCR werecarried out: (1) to detect and quantify the amountof transcript from each of the yeast helicase-likeORFs identified in this study; and (2) to examinethe expression profile of each ORF under variousphysiological conditions. Since a large scaleprofiling of gene expression allows us to deducebiological functions of the putative gene products(Naitou et al., 1997; Velculescu et al., 1997), weused seven different growth conditions to detectthe ORFs which have a unique expression profile:(1) vegetative growth of haploid S288C in a com-plete medium (SC) at 30)C; (2) vegetative growthof haploid S288C in a nutrient-starved medium


(SD) at 16)C; (3) heat shock during vegetativegrowth of haploid S288C; (4) sporulation, after thediploid strain RAY3A-D was maintained in avegetative growth condition; (5) UV irradiationduring vegetative growth of haploid S288C;vegetative growth of haploid S288C in the presenceof; (6) a DNA-damaging reagent (0·01% MMS); or(7) an inhibitor of DNA synthesis (50 m HU).The latter four conditions have been implicated inDNA repair, DNA recombination, and/or DNAreplication (Hampsey, 1997). The change in tran-scription level of each ORF in the seven conditionsis described in Table 2.

The results of Northern analysis of the 43helicase-like ORFs containing the 19 newly-identified ORFs, except for YMR128w and

Figure 4. Growth characteristics of the haploid null mutant ofsix non-essential ORFs. The haploid null mutant of each ORFwas incubated on a solid rich medium (YPAD). All of the plateswere carefully examined for signs of growth. Null mutants ofeach ORF were generated from two haploid strains, W303-1aand NOY396á. The results from the W303-1a strain are shownin this figure. When the indicated number of cells were spottedon the YPAD plate, the cells were then replaced on two plates,one of which was kept at 30)C and the other at 37)C, for twodays. The LP2915-9B (cdc9 mutant) and 185-3-4g (cdc28mutant) were used as positive controls for the ts phenotype. Itcan be seen that the growth rate of each ORF is different at30)C, as well as at 37)C. This demonstrates that the nullmutants of the ORFs (YDR332w, YGL064c, and YOL095c)are partially temperature-sensitive, and display the slow-growthphenotypes. The null mutants of YKL017c and YNL218w werenot examined.

Yeast 15, 219–253 (1999)

235 -

YNL218w, seven previously-characterized ORFswhose functions are unknown, and 17 helicase-likegenes whose functions are known, are summar-ized in Figure 5 and Table 2 (see ‘Materials andMethods’). It was found that the transcriptionallevel of all of the tested helicase-like ORFs is low(approximately 1/100–1/200 of the quantity ofACT1 mRNA). Each ORF has a unique band. Theobserved size of the transcript was longer than thatwhich would have been expected from the lengthof the actual ORF; this suggests the existence of anuntranslated region of a few hundred base pairs(see Table 2). Furthermore, the results from RT-PCR clearly indicate that the tested four ORFs,including YMR128w, are transcribed at low levelsin yeast cells (Figure 6). This is in agreement withthe previous observation by Rong and Klein(1993) that HPR5 is expressed at very low levels.These data indicate that all of the ORFs that weresubject to RT-PCR are transcriptionally active invegetatively growing cells.

Expression profiles of transcripts under variousconditions (Northern analysis)

The quantity of transcript encoded by 34 of the43 previously-characterized ORFs (79·1% of thetested ORFs) increased after heat shock treatment,as can be seen in lane 3 of Figure 5, and Table 2. Infact, upon heat shock treatment (39)C for 10 min),the quantity of transcript of the 34 ORFs increasedby 1·5- to 5·7-fold, as compared with the quantityof transcript under the vegetatively growing con-dition. Among the tested ORFs, the maximumincrease in transcript was seen in YHR169w, at5·7-fold. Transcriptional activation of helicase-related ORFs induced by heat shock, is fairlyvariable (1·5- to 5·7-fold), when compared withthat of the yeast ORF YFL016c, which has a7·1-fold increase upon heat shock treatment, andwhich is considered as a genuine heat shock-activated transcript (Naitou et al., 1997). Theobservation that the level of transcriptional acti-vation by heat shock, is variable among helicase-related proteins, is not due to a difference in theamount of total mRNA applied to the lanes, sincethe amount of RNA in each lane was adjustedprior to gel electrophoresis. Furthermore, theresults obtained from Northern analysis of fivecontrol ORFs done in our study (YFL007w,YFL039c [ACT1], YFL022c [FRS2], YFR050c[PRE4] and YFR052w [NIN1] are nearly identicalto the results obtained by Naitou et al. (1997) (see


Figure 5; data not shown on FRS2 and NIN1),excluding also the possibility described above.

On the other hand, a significant change in thequantity of transcript produced was not observedunder the other four conditions which were tested:nutrient starvation at low temperature (16)C);incubation in the sporulation medium; and treat-ment with either DNA alkylating reagent (0·01%MMS) or 50 m hydroxyurea (HU). It is knownthat unfinished replication caused by HU activatescell-cycle checkpoint signals; DNA damageinduced by UV and chemical agents also indepen-dently triggers this checkpoint mechanism(Elledge, 1996). Elledge et al. (1993), showed thatactivation of the checkpoint mechanism results inthe activation of damage-induced genes such asRNR2 and RNR3. This was confirmed in our study(see Figure 5; data not shown on RNR2). Whenyeast cells were treated with UV, DNA alkylatingreagent or HU, a 3·0-fold increase (mean of thethree conditions) in the transcript level of RNR2,and a 9·4-fold increase in that of RNR3, were seen.Although it is unclear whether MMS-induceddamage induces transcriptional activation like UVirradiation, the transcript levels of nine genes,including RAD16, increased in the presence ofHU. A general response to stress may cause theactivation of these genes.

It should be noted that the level of mRNA ofYER176w in the RAY3A-D strain markedlyincreased during sporulation by 3·1-fold. Over 30genes that are specific to sporulation in yeast, andwhich have a variety of functions, have beenreported. Genes that code the proteins involved inthe early phase of meiosis contain specific regulat-ory elements in their upstream sequence, forexample, the URS1 site (TCGGCGGCT), theUASH site (TGTGAAGTG) and the T4C site(TTTTCXXCG; X=any base), reported byMitchell (1994). Such regulatory elements were notobserved in the 5*-upstream region of YER176w,except for a potential UASH site at "148 bp(TGTGtAGTc). On the contrary, none of theseregulatory elements were identified in the ORFswhich encode proteins that are expressed in themiddle and late stages of meiosis. In this study,the mRNA level of YER176w was measured whenthe yeast cell was in the late stage of meiosis (i.e.after being cultured for 2 days in sporulationmedium). This suggests that YER176w could becategorized as a middle- or late-meiotic gene.Friedberg et al. (1991) reported that the expressionof several RAD genes is induced during meiosis, as

Yeast 15, 219–253 (1999)

Table entified ORFs and seven ORFs whose functions are unknown.

Descripteat shock)C, 10 min)

Sporulation(1% KAc, 2 days)

UV irradiation(120 J/m2)

MMS treatment(0·01% MMS)

HU treatment(50 mM HU)

ORFs w+a ++

+ +"

+ + "+

+ ++ +

+ + " +"

+ ++

+ ++ + + +

+ + ++ + ++

+ + ++ + ++ +

"+ + +

+ + + + + + ++ + + ++ + ++ + " +

236.

.

Copyright

?1999

JohnW

iley&

Sons,L

td.Y

east15,

219–253(1999)

2. Summary of Northern analysis which was performed on the 19 newly-id

ionORF name

(Gene name)

ORFsize(bp)

Observed sizeof transcript

(bp)

Nutrientstarvation

(16)C)H

(39

hose function is unknown (March 1998)YBR245c (ISW1) 3390 3400YDL031w (DBP10) 2897 3300YDL070w (BDF2) 1917 2200 +YDL084w (SUB2) 1341 1500YDR291w 3234 3300YDR332w 2070 2300YDR334w 4545 5200YFR038w 2337 2800YGL064c 1686 2000YGL150c (INO80) 4470 4700YHR031c 2172 2500YIR002c 2981 3000YKL017c (HCS1) 2052 2100YKL078w (JA2) 2208 2300YLR247c 4668 4700YLR276c (DBP9) 1785 1900YLR419w 4308 4400YOL095c (HMI1) 2126 2200YOR304w (ISW2) 3363 3400YGR271w 5918 6200YDL160c (DHH1) 1521 2300YER176w (ECM32) 3366 3300YHR169w (DBP8) 1296 1600YKR024c (DBP7) 2229 2500 +YMR290c (HAS1) 1517 1600 +YNR038w (DBP6) 1890 2100

237 -

Copyright ? 1

Tab

le2.

Con

tinu

ed.

Des

crip

tion

OR

Fna

me

(Gen

ena

me)

OR

Fsi

ze(b

p)

Obs

erve

dsi

zeof

tran

scri

pt(b

p)

Nut

rien

tst

arva

tion

(16)

C)

Hea

tsh

ock

(39)

C,

10m

in)

Spor

ulat

ion

(1%

KA

c,2

days

)U

Vir

radi

atio

n(1

20J/

m2 )

MM

Str

eatm

ent

(0·0

1%M

MS)

HU

trea

tmen

t(5

0m

MH

U)

OR

Fs

who

sefu

ncti

onis

know

nD

NA

repa

irY

BR

114w

(RA

D16

)23

7325

00+

++

++

++

++

+Y

ER

171w

(RA

D3)

2337

2500

++

++

+(+

reco

mbi

nati

on)

YG

L16

3c(R

AD

54)

2697

2900

++

+(+

tran

scri

ptio

n)Y

IL14

3c(S

SL

2)25

3227

00+

++

++

Pre

-mR

NA

splic

ing

YB

R23

7w(P

RP

5)25

5026

00+

+Y

NR

011c

(PR

P2)

2628

2700

++

YE

R01

3w(P

RP

22)

3435

3500

++

++

YG

L12

0c(P

RP

43)

2304

2500

"+

+"

rRN

Apr

oces

sing

YD

R02

1w(F

AL

1)22

0015

50+

+Y

GL

078c

(DB

P3)

1572

1700

++

++

++

+Y

GL

171w

(RO

K1)

1695

1800

++

+Y

JL03

3w(H

CA

4)23

1324

00+

++

++

mR

NA

tran

spor

tY

JL05

0w(M

TR

4)32

2233

00+

++

Mit

ocho

ndri

alfu

ncti

onY

ML

061c

(PIF

1)25

8028

00+

Chr

omos

ome

segr

egat

ion

YB

R07

3w(R

DH

54)

2876

2900

"+

+T

rans

crip

tion

alre

gula

tion

YE

R16

4w(C

HD

1)44

0646

00D

NA

repl

icat

ion

YG

L20

1c(M

CM

6)30

5432

00+

"

Con

trol

sA

bund

ant

tran

scri

ptY

FL

039c

(AC

T1)

1437

1500

"H

eat

shoc

k-sp

ecifi

ctr

ansc

ript

YF

L01

6c15

3316

50+

++

++

DN

Ada

mag

e-in

duce

dtr

ansc

ript

YIL

066c

(RN

R3)

2610

2900

++

++

++

++

++

++

++

++

Spor

ulat

ion-

spec

ific

tran

scri

ptY

FR

032c

867

1000

++

++

Con

trol

tran

scri

pts

from

Chr

.V

IO

RF

sY

FL

007w

5412

6400

+Y

FR

050c

(PR

E4)

798

800

"+

a Eva

luat

ion

ofth

ele

vel

ofea

chhy

brid

ized

sign

alw

asca

rrie

dou

tas

follo

ws:

for

nutr

ient

star

vati

on,

heat

shoc

k,an

dsp

orul

atio

n,th

ere

lati

vele

vel

ofea

chsi

gnal

was

calc

ulat

edre

lati

veto

the

tran

scri

ptle

vel

ofth

eve

geta

tive

grow

thco

ndit

ion

(lan

e1

inF

igur

e5)

,w

hich

was

assi

gned

ava

lue

of1·

0.F

orU

Vir

radi

atio

nan

dth

edr

ugtr

eatm

ents

(MM

San

dH

U),

the

rela

tive

leve

lof

each

ofth

ese

sign

als

was

calc

ulat

ed,a

ssig

ning

the

resp

ecti

vele

velo

fco

ntro

ltra

nscr

ipt

(i.e

.lan

es5

and

7in

Fig

ure

5),a

valu

eof

1·0.

The

abun

danc

eof

the

sign

alre

lati

veto

aco

ntro

l(w

hich

was

assi

gned

ava

lue

of1·

0)w

asev

alua

ted

asfo

llow

s:"

,red

uced

leve

l(le

ssth

an0·

5-fo

ld);

+,i

nduc

ed(1

·5–2

·0-f

old)

;+

+,i

nduc

ed(2

·0–3

·0-f

old)

;+

++

,in

duce

d(3

·0–4

·0-f

old)

;+

++

+,

high

lyin

duce

d(m

ore

than

4·0-

fold

),re

spec

tive

ly.

999 John Wiley & Sons, Ltd. Yeast 15, 219–253 (1999)


239 -

well as by UV damage. It has also been reportedthat two helicase-related genes, RAD16 andRAD54, are transcriptionally activated during theearly stage of meiotic events (Cole et al., 1989;Bang et al., 1995). Failure in detecting increasedlevels of the mRNA of both RAD16 and RAD54 inour samples also suggests that induction of thetranscription of YER176w is a late-meiotic event(see Table 2).

Furthermore, transcription of eight out of the 43examined ORFs (18·6%) was repressed duringsporulation. This is probably a general phenom-enon because the level of expression of 30 out ofthe 104 ORFs on chromosome VI (28·8%) wasrepressed to less than 50% of the normal levelsunder the sporulation condition in our previousstudy (Naitou et al., 1997).

Finally, YFR038w and YKL017c contain cellcycle-dependent transcription factors, which were


located at "668 bp (ACGCGTGA for Mbf1p),and at "797 bp (TTTTCGTG for Sbfp), respect-ively, in the 5*-upstream sequence of the respectiveORFs (Dhawale and Lane, 1993). This suggeststhat the expression of these two ORFs may dependon the cell cycle.

DISCUSSION

Helicase-like genes in Saccharomyces cerevisiaeIn this study, we have identified 134 helicase-like

ORFs in the yeast genome and systematicallycharacterized more than 21 novel ORFs by genedisruption and Northern analysis. For screeningthe helicase-like sequences, we used the amino acidsequences of authentic yeast helicase or helicase-family proteins as probes for ‘in silico’ hybridiz-ation. Here, we must discuss two issues: one is theexistence of pseudo-positive sequences in the iso-lated ORFs. For example, Snf2p and Sth1p con-tain sequences that are typically seen in proteinswhich make up the DExH-box family, in additionto transcriptional activation domain with bromo-domain (PROSITE number PS00633; Jeanmouginet al., 1997), an amino acid sequence that isevolutionarily conserved in several proteins andwhich is involved in transcriptional regulation. Sixproteins in Cluster II and GCN5 that have themost similarity with both authentic proteins (i.e.the BLAST p-value for each of the seven proteinsis less than 1·0#e"5) were identified; however,five out of these six ORFs contain the bromo-domain, but not any helicase motifs. Although it isquestionable whether these seven proteins exhibithelicase activity, since they lack NTP-bindingmotifs, they were placed among ‘the helicase-likeORFs’ in this study.

The second issue is related to the Sug1p-likeproteins of the AAA superfamily. The Sug1pencoded by YGL048c is a protein which has beenconserved from yeast to humans. This protein hasbeen shown to function in protein degradation asa component of the 26S proteasome in various

Figure 6. RT-PCR which was performed on four helicase-likeORFs whose transcription could not be detected by Northernanalysis. The transcripts of four ORFs (YJL092w [HPR5],YJR035w [RAD26], YLR032w [RAD5], and YMR128w),which could not be detected by Northern analysis, weredetected by RT-PCR. In each lane, the reverse-transcriptionreaction was performed in the presence (+) or absence (") ofreverse transcriptase and the mRNA sample, and then PCRwas carried out using the reaction mixture and ORF-specificprimers. The expected size of the products are: YJL092w,500 bp; YJR035w, 500 bp; YLR032w, 500 bp; YMR128w,504 bp; YFL039c (ACT1) which was used as the positivecontrol, 327 bp. M, molecular weight markers (mixture ofDNA fragments of Dra I, and DNA fragments from HindIII-and MspI-digested pUC19).

Figure 5. Systematic Northern analysis of 43 yeast helicase-like ORFs. For each ORF, the pattern of Northern hybridization isshown. Haploid S288C cells were used in all of the conditions except for the sporulation condition. Total RNA (30 ìg per lane) wasprepared from yeast cells that had been cultured under the following conditions: lane 1, vegetative growth; lane 2, nutrientstarvation at 16)C; lane 3, heat shock; lane 4, diploid RAY3A-D cells under sporulation condition; lane 5, negative control for UVirradiation; lane 6, UV irradiation; lane 7, negative control for the MMS and HU treatments; lane 8, MMS treatment; lane 9, HUtreatment. The 17 helicase-like ORFs in the upper part of the right column of the figure have known functions. The ORFs onchromosome VI were used as the control for each condition in the lower part of the right column and the typical figure of the gelstained with ethidium bromide was also shown in the bottom of the right column.

Yeast 15, 219–253 (1999)

240 . .

eukaryotes (Baumeister et al., 1998). Recently,Fraser et al. (1997) reported that mouse SUG1protein exhibits both ATPase and DNA helicase(3*]5*) activity and Weeda et al. (1997) found theassociation of SUG1 protein with the XPB subunitof TFIIH. Makino et al. (1997) reported thatATPase activity of the SUG1 protein of the rat canbe activated by specific RNAs. Conservation ofsome of the helicase-specific motifs among SUG1proteins has been also reported (Fraser et al., 1997;Weeda et al., 1997). These data suggest that theSug1p protein in yeast may exhibit either nucleicacid-dependent ATPase or DNA helicase activity,although there have been no reports of helicaseactivity in yeast Sug1p.

More than 20 yeast ORFs that have a predomi-nant similarity to the ORF which encodes Sug1pwere identified in the screening. These sequencesshare the helicase motif VI-like domain in additionto the Walker NTP-binding motifs A and B, butdo not contain any domains with similarity toother helicase-specific motifs (data not shown).Some of these ORFs have been identified toencode putative ATPase subunits of the 26S pro-teasome (Schnall et al., 1994). These ORFs encodethe proteins that belong to the so-called ‘AAA(ATPases Associated to a Variety of CellularActivities) superfamily’ of proteins; Sug1p is also amember of the AAA superfamily (Confalonieriand Duguet, 1995). Among the roles that proteinsof the AAA superfamily play are as ATP-dependent molecular chaperones in proteolysis,membrane fusion and transport for secretion andendocytosis, etc. These functions do not seem to berelated to nucleic acid metabolism. However,Confalonieri and Duguet (1995) suggested thatsome of the proteins of the AAA family undergo aconformational change upon ATP-binding and/orATP hydrolysis. This suggests that some of theproteasome subunits of an ATPase on a protein ofthe AAA family can use the energy generated byATP hydrolysis for disassembling and unfoldingsubstrate proteins. This schema is similar to themolecular mechanism of DNA/RNA unwindingactions by helicases, except that the substrate isdifferent (Lohman and Bjornson, 1996). That is,helicases unwind double-stranded DNA or RNAto a single-stranded template; the ATPase subunitsof a proteasome may catalyse the unfolding ofsubstrate proteins into polypeptide chains. Thus,Sug1p or other members of the AAA familymay play a role in nucleic acid metabolism inSaccharomyces cerevisiae.


Relationship between sequence similarity andbiological function in helicase-like proteins

In phylogenetic analysis, we found that theproteins exhibiting sequence similarities to heli-cases are widely diverged to make up severalsubfamilies (up to 11), and observed close relation-ship between the subfamilies (i.e. sequence simi-larities) and their biological functions, which areindicated by colour boxes in Figure 2. Forinstance, the proteins in Cluster II have beenreported as being part of the RSC (Remodel theStructure of Chromatin) and SAGA (Spt-Ada-Gcn5-Acetyltransferase) complexes, which act aschromatin remodelling machines (Kadonaga,1998), and the members which make up ClustersIII and IX are related to minichromosome main-tenance (MCM) and replication factor C (RFC),which are involved in the initiation and elongationof cellular DNA replication (Chong et al., 1996;Baker and Bell, 1998). The ORFs which make upClusters I and XI encode many SNF/RAD-likeproteins; many of these proteins are involved inDNA repair, recombination, chromatin remodel-ing and transcriptional activation (Eisen et al.,1995). Most of the ORFs which make up ClusterVII encode proteins of the DEAD family; many ofthese proteins are involved in RNA-related cellularprocesses, for example, rRNA synthesis, trans-lation and pre-mRNA splicing, as depicted inFigure 2 (indicated by yellow, pink, and red boxes,respectively). Cluster X contains four PRP pro-teins, which have been reported to be involved inpre-mRNA splicing (Staley and Guthrie, 1998).

In addition, the proteins encoded by the ORFsin Cluster V (DExxQ family) should be noted. Ingeneral, not all of the proteins containing typicalhelicase motifs exhibit nucleic acid-dependentATPase and/or ATP-dependent helicase activity(see Table 1). However, four of the five proteinswhich make up Cluster V (i.e. except for Sen1p)interestingly exhibit both nucleic acid-dependentATPase and ATP-dependent DNA/RNA helicaseactivity [Dna2p (Budd and Campbell, 1995),Nam7p (Upf1p; Czaplinski et al., 1995), Hel1p(YER176wp; Bean and Matson, 1997), and Hcs1p(YKL017cp, Biswas et al., 1997)].

Disruption of helicase-like ORFsSystematic gene disruption of each of the 21

newly-identified ORFs was carried out, since alarge-scale phenotypic analysis of null mutants isuseful for clarifying biological functions of the

Yeast 15, 219–253 (1999)

a

Predictedtransmembrane

helices(TMpred)

Predicted cellularlocalization

(PSORT score)c

Phenotype ofnull mutant

(haploid)

2 None Viable

1 Nucleus (0·96) Viable1 Nucleus (0·98) Viable

1 Nucleus (0·73) Viable1 Nucleus (0·97) Lethal1 Nucleus (0·76) Viable

None Nucleus (0·98) Lethal

None Nucleus (0·98) Viable

1 None Viable2 Mitochondrial matrix space (0·89) Slow growth

1 Nucleus (0·96) ViableNone Nucleus (0·96) LethalNone None Lethal

None None LethalNone None Slow growthNone None Viable

2 None Lethal

1 Nucleus (0·98) Lethal

2 Plasma membrane (0·70) Viable

None None Slow growth1 Endoplasmic reticulum (0·85), nucleus (0·82) Viable

nd characterization of the proteins by these ORFs, were carried out.

ORFs which have a PSORT score of greater than 0·70.

241

-

Copyright

?1999

JohnW

iley&

Sons,L

td.Y

east15,

219–253(1999)

Table 3. Characteristics of the 21 newly-identified helicase-like ORFs .

ORF name(Gene name) Subcluster Chr.

Length(amino acids) Features of proteinb

YBR245c (ISW1) I.1 II 1129 Strong similarity to SNF2/SWI2 DNA binding regulatory protein,SANT domain also found in Swi3p, Ada2p, Tfc5p and Rsc8p

YOR304w (ISW2) I.1 XV 1120 Strong similarity to human Snf2p homologueYDR334w I.3 IV 1514 Similarity to nuclear Sth1p, Snf2p and related proteins

ATP-dependent DNA ligase signature (PS00697)YFR038w I.3 VI 778 Strong similarity to mouse lymphocyte specific helicaseYGL150c (INO80) I.3 VII 1489 Similarity to Snf2p and human SNF2á, two ATP-binding sitesYHR031c I.7 VIII 723 Similarity to Pif1pYDL070w (BDF2) II.2 IV 638 Similarity to bromodomain protein Bdf1p, bromodomain signature

(PS00633)YKL017c (HCS1) V.1 XI 683 DNA helicase A which is stimulated by RPA, interaction with

DNA polymerase áYDR291w VI.2 IV 1077 Similarity to prokaryotic RNA helicasesYDR332w VI.3 IV 689 Weak similarity to RNA helicase Mss116p, Glu/Leu/Phe/Val

dehydrogenases active site (PS00074)YIR002c VI.3 IX 993 Weak similarity to ATP-dependent RNA helicasesYDL031w (DBP10) VII.1 IV 995 DEAD-box ATP-dependent helicase familyYDL084w (SUB2) VII.1 IV 446 61% Identity to human UAP56 protein, which is required for

association of U2 snRNP with the pre-spliceosoneYLR276c (DBP9) VII.4 XII 594 DEAD-box ATP-dependent helicase familyYGL064c VII.6 VII 561 DEAD-box ATP-dependent helicase familyYNL218w IX.4 XIV 587 Similarity to E. coli DNA polymerase III subunits (ã and ô)YKL078w (JA2) X.1 XI 735 Strong similarity to ATP-dependent RNA helicases, two

ATP-binding sitesYMR128w (ECM16) X.1 XIII 1267 DEAH-box ATP-dependent RNA helicase signature, cell wall and

membrane defects by Tn3 insertionYLR419w X.2 XII 1435 Similarity to cattle DNA helicase II and human RNA helicase A,

Asp/Glu racemases signature (PS00924)YOL095c (HMI1) XI.1 XV 706 Similarity to prokaryotic helicasesYLR247c XI.2 XI 1556 Similarity to S. pombe and rad8 protein and Rdh54p, EF-hand

calcium-binding domain (PS00018), zinc finger (C3HC4 type)(PS00518)

aThe phenotypes of the null mutants of these ORFs, have not been reported (as of March 1998). Systematic gene disruption abFeatures of the proteins were obtained from the public databases (MIPS, YPD and SGD).cPredicted cellular localization of the protein encoded by an ORF. The predicted cellular localization was only determined for

242 . .

Co
pyright ? 1999 John Wiley & Sons, Ltd. Yeast 15, 219–253 (1999)

243 -

protein encoded by the disrupted ORFs (Riegeret al., 1997). The seven newly-discovered yeasthelicase-like ORFs that were found to be essentialwere classified based on similarity in amino acidsequence to known proteins (Figure 2): YGL150c(SNF2 family, Cluster I); YDL070w (RSC family,Cluster II); YDL031w, YDL084w and YLR276c(DEAD family, Cluster VII); YKL078w andYMR128w (PRP family, Cluster X).

In general, null mutations of the following genesare lethal: genes which encode proteins involved inDNA replication (for example, the MCM andRFC proteins in Clusters III and IX); rRNAprocessing (numerous DEAD-box proteins inCluster VII); and pre-mRNA splicing (PRP pro-teins in Cluster X) (see Table 1). As describedabove, most of these essential ORFs (five out ofseven) belong to Cluster VII, which encode pro-teins of the DEAD family, and Cluster X, whichencode proteins of the PRP protein family. Bycontrast, most of the dispensable ORFs encodeproteins that are involved in DNA repair andrecombination (e.g. many of the RAD proteins inCluster I) and transcriptional regulation via chro-matin remodelling (e.g. the Snf2p-like proteins, orproteins which make up the RSC complex inClusters I and II). Six of the 21 newly-identifiedORFs classified to Cluster I; of these, five werefound to be dispensable.

Essential helicase-like ORFsThe biological features of the proteins encoded

by newly-identified 21 ORFs are summarized inTable 3. Structural features of the proteinsencoded by these ORFs are illustrated in Figure 7.Each of the seven essential yeast helicase-likeORFs encodes the putative polypetide describedbelow:

YGL150c This is the sole essential yeast helicase-like ORF out of the six ORFs in Cluster I. It


exhibits similarity to ORFs that encode proteins ofthe SNF/RAD-family. A BEAUTY search foundthat the amino acid sequence of the proteinencoded by YGL150c has strong similarity withthe protein sequences of global transcriptionalactivators; the protein encoded by YGL150c hasstrong similarity with the SNF2 protein in humansand the BRM proteins in Drosophila. The PSORTprogram predicted that the protein encoded byYGL150c is a nuclear protein (Table 3). Thissuggests that this protein may play an importantrole in transcriptional activation. The part of theORF which encodes the half which contains theamino terminus of the protein is composed ofa unique sequence with an ATP-binding site.However, the functional role of the proteinencoded by this ORF is unclear. (Note: recently,i.e. during submission of this paper, YGL150c hasbeen named ‘INO80’ due to ‘inositol deficientphenotype’ in SGD.)

YDL070w YDL070w was classified into ClusterII, which contains ORFs that encode proteins ofthe RSC complex. YDL070w exhibits significanthomology (a BLAST p-value of 1·0#e"96) to theyeast ORF encoding Bdf1p. Bdf1p is a chromo-somal component, and is involved in sporulation(Chua and Roeder, 1995). It is unlikely that theprotein encoded by YDL070w has helicase activitybecause of the absence of helicase-related motifs(Figure 7). This protein may, however, be involvedin transcriptional activation via chromatin re-modeling. The protein encoded by YDL070w mayplay a more essential role in transcripton thanother yeast proteins that are involved in remodel-ling (e.g. Rsc1p or Rsc2p), since the ydl070w nullmutation was lethal. (Note: YDL070w has beennamed ‘BDF2’.)

YDL031w The putative protein encoded byYDL031w contains seven helicase-conserved

Figure 7. Structural features of the putative protein encoded by each of the 21 newly identified helicase-like ORFs. Each ORF wassubject to gene disruption. Of the putative proteins encoded by the 21 ORFs, six ORFs classified to Cluster I; three ORFs to ClusterVI; four ORFs to Cluster VII; three ORFs to Cluster X; and one ORF each to Clusters II, V, IX and XI. The presence of motifsand protein signatures in each putative protein is indicated by the colour symbols described in the figure. The seven helicase motifs(I–VI, as well as Ia), are indicated at the top of the figure. The presence of particular motifs in the putative proteins is indicatedby a green box. The TMpred program (Hofmann and Stoffel, 1993), was used to predict the presence of membrane-spanningsegments in each protein; the location of the membrane-spanning segments are indicated by a yellow box in the figure. Otherdomains in the putative proteins, such as ‘DEAD’ in motif II, are also indicated. The SNF2-like domains in each protein encodedby the ORFs which classified to Cluster I, are shown in green. YNL218w contains one SRC box, which share with the proteins ofRFC family (Cullman et al., 1995). The ORFs whose ORF name is encompassed by a red box in the figure are essential forvegetative cell growth under normal conditions; the ORFs whose ORF name is encompassed by a blue box are not essential forvegetative cell growth.

Yeast 15, 219–253 (1999)

244 . .

motifs, including the DEAD-box, as well as sevenputative nuclear targeting signals (see Figure 7).The part of the ORF which encodes the carboxylterminal half of this protein has weak homologywith proteins of the band 4·1 family; the rest of thesequence in this half of the protein is fairly unique.Proteins which make up the band 4·1 family playcritical roles in regulating the interaction betweenthe cytoskeleton and plasma membrane (Takeuchiet al., 1994). (Note: recently, YDL031w has beennamed ‘DBP10’, i.e. ‘DEAD box protein 10’, inSGD.)

YDL084w The putative protein encoded byYDL084w contains the seven conserved motifs ofhelicases; motif II includes a modification of theDEAD-box and the DECD. YDL084w does notcontain nuclear targeting sequences. The encodedprotein is 61% identical to the UAP56 protein inhumans. The UAP56 protein is a DECD proteinthat is required for bringing together U2 snRNPwith the pre-spliceosome in the pre-mRNA splic-ing pathway (Staley and Guthrie, 1998). TheUAP56 protein is recruited to the pre-mRNA bythe protein U2AF65. The UAP56 protein itself isrequired for the U2 snRNP-branchpoint inter-action (Fleckner et al., 1997). This gene is also asuppressor of the cold-sensitive snRNP biogenesismutant, brr-1-1 (Staley and Guthrie, 1998). Thesestrongly suggest that Ydl084wp is the yeasthomologue of the UAP56 protein in humans, andthat Ydl084wp is involved in pre-mRNA splicing.This putative function is consistent with lethalityof the ydl084w null mutation. (Note: YDL084whas been named ‘SUB2’.)

YLR276c YLR276c is classified to Cluster VII.The protein encoded by YLR276c belongs to theDEAD family, as do the proteins encoded byYDL031w and YDL084w. Although YLR276chas previously been named ‘DBP9’ (DEAD-boxprotein 9) by Daugeron (SGD), genetic and bio-chemical information on the protein encoded bythis ORF, has not been reported. The results fromour study indicate that Dbp9p is essential for cellgrowth, although its cellular function remains tobe determined.

YKL078w The protein encoded by this ORF,which was named ‘JA2’, has seven conservedmotifs in addition to an extra NTP-binding loop atamino acid numbers 413–420. The amino acidsequence of the protein encoded by YKL078w is


similar to the amino acid sequences of PRP pro-teins. PRP proteins are involved in pre-mRNAsplicing, suggesting that the protein encoded byYKL078w may also be involved in pre-mRNAsplicing.

YMR128w This ORF has previously been named‘ECM16’ (Extra-Cellular Mutant) by Lussier et al.(1997). Lussier et al. (1997) reported that Tn3insertion into the yeast ORF YMR128w, usingthe E. coli system, causes the Tn-inserted yeastmutants to become hypersensitive to a cell-surface-polymer perturbing agent (i.e. calcofluor white).This seems to suggest that YMR128w is notessential for cell viability. According to ourinvestigations, however, this is incorrect. In fact,in their systematic screening for genes that areinvolved in the biosynthesis and architecture of thecell surface of yeast cells, Lussier et al. (1997)found the Tn insertions into three helicase-likeORFs, YER176w (ECM32), YJL033w (HCA4)and YMR128w (ECM16). It has recently beenshown that YJL033w encodes an essential helicase-like protein which functions in rRNA synthesis(Liang et al., 1997). It is interesting to note thatmutations in these three helicase-like ORFs pheno-typically lead to defects in the cell wall of yeastcells. However, it is unlikely that the proteinsencoded by these three ORFs, including ECM32which has been reported to contain DNA helicaseI by Bean and Matson (1997), are involved inmembrane functions, in addition to their putativenuclear functions. Regarding the putative proteinencoded by YMR128w, bioinformatical analysessuggest that it locates in nuclei, and that it hasbeen evolutionarily conserved based on the identi-fication of several putative homologues (or ana-logues) in the genomes of S. pombe (prh1 protein,S62466). C. elegans (C06E1.10 protein, P34305)and Drosophila (62D9.b protein, AL009171).

Non-essential helicase-like ORFsWe also identified 14 non-essential ORFs and

three haploid null mutants (YDR332w, YGL064cand YOL095c) exhibited slow-growth phenotypes(Figure 4). Baganz et al. (1997) reported that thechoice of a nutritional marker for gene disruptionmay affect the growth phenotypes; however, theseresults suggest that the proteins encoded by theseORFs play important roles in primary cellularprocesses, such as global transcriptional acti-vation, structural modification of the chromosome

Yeast 15, 219–253 (1999)

YLR357w (RSC2) in block #30; YDL070w andYLR399c (BDF1) in block #15; and YOR204w

245 -

and RNA metabolism. In fact, yeast proteins ofthe RSC complex are thought to be involved inchromatin remodeling; yeast proteins of theSAGA complex are involved in the transcriptionalactivation of several genes (Razin and Kadonaga,1997; Kadonaga, 1998). Null mutations of geneswhich encode particular components of both theRSC and SAGA complexes (i.e. RSC1 and RSC2;GCN5 and SPT7) lead to a slow-growth pheno-type. It has also been reported that the nullmutations of DBP3 (which functions in rRNAprocessing), as well as the null mutation of CTF18(which functions in chromosome segregation),cause slow growth (Weaver et al., 1997; Kouprinaet al., 1994).

On the other hand, a null mutation in each ofnine ORFs (except for YKL017c and YNL218w)does not affect cell growth. Two possible ideascould account for this phenotype. First, the func-tion of each of the nine ORFs may not be essentialfor mitotic cell growth under the growth con-ditions we used in our study. In fact, numerousmutants that have a null mutation of a geneinvolved in DNA repair and recombination (e.g.RAD54 and RAD16), are healthy in regard to cellgrowth but exhibit various defects in repair andrecombination functions upon DNA damage andin meiosis (see Table 1). It can be deduced that thefunctions of each of these nine ORFs are notneeded in the mitotic cell cycle. Furthermore, itcan be proposed that a null mutation in ORFswhich encode proteins involved in the transcrip-tional regulation of genes that are not essential fornormal cell growth, are also not needed for cellviability.

Another idea is that each of these nine ORFsmay be complementary in function with anothergene. Linder and Slonimski (1989) reported thatTIF1 and TIF2, translation initiation factors whichboth have RNA helicase activity, are structurallyand functionally identical, and that a null mu-tation in one gene does not affect the viability ofthe cell. This dispensability could be due to comp-lementation by the presence of a similar ORF,which has been part of a duplicated chromosomalregion—the loss of function of one gene can becompleted by the function of the other intact gene.In the yeast genome, 55 blocks of duplicatedchromosomal regions have been identified byWolfe and Schields (1997); among these, four pairsof helicase-like genes, including TIF1 and TIF2,were detected: YKR059w (TIF1) and YJL138c(TIF2) in block #41; YGR056w (RSC1) and


(DED1) and YPL119c (DBP1) in block #51. How-ever, of the 21 newly identified ORFs, putativehomologues in yeast of the nine ORFs which arenot essential for cell viability could not be found inthe corresponding chromosomal blocks. Further-more, the presence of a duplicated gene does notnecessarily indicate that the loss of function of oneof the genes is always made up by the other gene.In other words, two proteins, one in each of theduplicated chromosomal regions, are not necess-arily functionally identical. Indeed, the resultsfrom our study indicate that this theory is notapplicable to all proteins in a chromosomal regionwhich has been duplicated. In our study we foundthat a null mutation in YDL070w was lethal, eventhough its intact partner, YLR399c (BDF1), waspresent. A mutation in DED1 also leads to a lethalphenotype, even in the presence of the respectiveintact partner (Chang et al., 1997). These areunlike the case of the TIF genes, TIF1 and TIF2.Thus, the dispensability of these nine genes arelikely accounted by the former idea.

Regarding the putative function of the 14proteins encoded by these non-essential ORFs,we used a bioinformatical approach to look forevidence which may suggest the biological functionof these proteins (see Figure 7 and Table 3).

Cluster I proteins The protein encoded byYBR245c contains a SANT domain, which hasbeen conserved in several co-activators includingSwi3p, Ada2p, etc. (Aasland et al., 1996). Thisindicates that Ybr245cp could be a transcriptionalregulator. A BEAUTY search indicated that theputative proteins encoded by YBR245c, YFR038wand YOR304w contain a core region (an approxi-mately 500-amino acid stretch) that is presentamong proteins of the SNF2 family. The putativeproteins encoded by YGL150c and YOR304wcontain a similar core domain; however, anapproximately 250–500 amino-acid stretch ofunique sequence is inserted into the core domain(see the SNF2-like regions which are denoted ingreen in Figure 7). The three putative proteinsencoded by YBR245c, YOR304w and YDR334wcontain the leucine zipper motif. This indi-cates that these proteins may be able to forma multiprotein complex with other leucinezipper-containing proteins.

The human ATRX protein belongs to the SNF2family. Defects in the ATRX protein lead to an

Yeast 15, 219–253 (1999)

246 . .

ATR-X syndrome (for example, á-thalassemia,genital abnormalities, some types of mentalretardation). Gibbons et al. (1995) suggested thatthe ATRX protein is involved in the transcrip-tional regulation of other genes, including theá-globin gene. In addition to the helicase-likedomain, the amino acid sequence of the ATRXprotein contains a PHD finger-like domain with aunique C4HC3-type zinc finger; this particularsequence in yeast has been implicated inchromatin-mediated transcriptional control(Aasland et al., 1995). These data suggest that thehuman ATRX protein is involved in transcrip-tional regulation, possibly through chromatinremodelling. The human ATRX protein is a mem-ber of the SNF2 family. Yeast helicase-like ORFsthat exhibit a similarity to proteins of the SNF2family, classified to Cluster I. It would be interest-ing to characterize homologues of the Cluster Iyeast helicase-like ORFs, in higher eukaryotes.(Note: recently, YBR245c and YOR304w havebeen named ‘ISW1’ and ‘ISW2’, respectively.)

Cluster V protein The protein encoded byYKL017c has previously been named HCS1.Biswas et al. (1997) reported the Hcs1p to be DNAhelicase A, and that this helicase is dependent onreplication protein A (RPA) and DNA polymeraseá. This suggests that the protein encoded byYKL017c may play a major role in cellular DNAreplication, similar to Dna2 helicase (Yhr164cp;Budd and Campbell, 1995). In general, replicativeDNA helicases, such as the DnaB proteins in E.coli and Dna2p in yeast, are essential for cellgrowth; however, we showed that YKL017c isdispensable for cell viability. Furthermore, Ishimi(1997) reported that the complex of MCM4,MCM6 and MCM7, members of the humanMCM protein, are associated with DNA helicaseactivity, and proposed that the multimeric form ofthese MCM proteins is a major replicative DNAhelicase. The ORFs which encode yeast MCM-related proteins, are classified to Cluster III (seeFigure 2), and the MCM proteins in yeast areessential components of the pre-replication com-plex of yeast (Baker and Bell, 1998). These indicatethat the protein encoded by YKL017c unlikely actsas the major DNA helicase for DNA replication inyeast. However, it is still quite possible thatYk1017cp is involved in DNA replication, in thatit could be functionally similar to PriA helicase inE. coli. The PriA helicase is dispensable, and isinvolved in damage-induced DNA replication, as


well as in homologous recombination and double-strand break repair in E. coli (Kogoma et al.,1996).

Cluster VI proteins Information is limited onthree yeast helicase-like ORFs that classified toCluster VI and that were found to be dispensable.The putative protein encoded by YDR332w is abasic protein (pI 9·1), whose amino acid sequencecontains the Glu/Leu/Phe/Val dehydrogenases sig-nature of four kinds of NAD-dependent reversibledeaminases. The null mutant of YDR332w exhib-its the slow-growth phenotype. The putative pro-tein encoded by YDR332w has similarity with typeI restriction enzymes, the UvrB subunit of theUvrABC excinuclease in E. coli, and yeast RNAhelicases. The putative cellular localization of theprotein encoded by YDR332w is the mito-chondrial matrix space, which suggests that thefunction of this protein in yeast may be to processnucleic acids in the mitochondria.

Less information could be found to deduce thepossible function of the proteins encoded byYIR002c and YDR291w. The sequences of both ofthese helicase-related proteins are similar to anumber of RNA helicases. The DNA sequence ofthe non-helicase domains in both of these ORFs isunique.

Cluster VII protein Out of the four DEAD-boxcontaining ORFs that were newly identified,YGL064c was the sole ORF that was not essentialfor mitotic cell growth. However, the null mutantof this highly basic protein (pI 10·1) exhibited theslow-growth phenotype, suggesting the functionalimportance of this protein.

Cluster IX protein The putative protein encodedby YNL218w has similarity to E. coli DNA pol-ymerase III subunits ã and ô and several RFCsubunits. These proteins are essential and playroles in DNA chain elongation; however, itis unlikely that Ynl218wp functions in DNAreplication because this ORF is dispensable.

Cluster X proteins The putative protein encodedby YLR419w has a molecular weight of 163·1 kDaand contains the signature of aspartate andglutamate racemase, which is required for thebiosynthesis of peptideglycan (PS00924). Theamino-terminal region of the protein encoded byYLR419w, is unique; however, it has a weak

Yeast 15, 219–253 (1999)

247 -

similarity to the rod-like tails of Uso1p (P25386),an intracellular transport protein in yeast, and themyosin-1 isoform (P08964).

Cluster XI proteins The null mutant of YOL095cdisplays the slow-growth phenotype. The DNAhelicases of prokaryotes, such as the PcrA proteinof Bacillus, which is essential for cell viability, andboth the UvrD and Rep helicases of E. coli, whichare involved in DNA repair (Matson and Kaiser-Rogers, 1990), contain similarities to the aminoacid sequence of the protein encoded by YOL095c.The yeast ORF with the highest similarity toYOL095c is YJL092w (HPR5), which encodes aDNA helicase for DNA repair and recombination.These evidence suggest that the protein encoded byYOL095c may play a role in DNA repair and/orrecombination. The protein encoded by YLR247chas a RING-type zinc finger, which would allowthe protein to interact directly with DNA. Theseproteins whose amino acid sequence contains aRING-type zinc finger and/or a leucine zippermotif, may play a role in either transcriptionalregulation or DNA reair. (Note: recently,YOL095c has been named ‘HMI1’ in SGD.)

Transcriptional activation of helicase-like genes byheat shock

In this study, we have observed an interestingtranscriptional activation of helicase-related genesby heat-shock treatment (Figure 5). This phenom-enon can be explained as follows. It has beenreported that heat shock causes transcriptionaland translational exchanges in both Saccharo-myces cerevisiae and Drosophila (Lindquist, 1981;DiDomenico et al., 1982). In yeast, the half-life ofmRNA is typically measured in minutes (Chia andMcLaughlin, 1979) and, upon heat shock, mRNAsare rapidly degraded and disappear from the cells,leaving the cells free to shift to synthesizingheat-shock proteins. When yeast cells shift fromsynthesizing non-heat-shock proteins to the syn-thesis of heat-shock proteins, it is reasonable toassume that genes involved in translation, rRNAsynthesis and pre-mRNA splicing are transcrip-tionally activated, and that the levels of theseproteins are themselves increased to compensatefor the lack of synthesis of non-heat shock pro-teins. The induction of the transcription of PRPgenes, which include FAL1, DBP3, ROK1 andHCA4, could apply to this situation. Craig (1992)reported that three glycolytic enzymes (i.e. enolase,


glyceraldehyde-3-phosphate dehydrogenase andphosphoglycerate kinase) are inducible upon heatshock to cope with the increased energy demand.This may be similar to the situation describedabove.

Upon heat-shock treatment, we also observedelevated mRNA levels of repair-related genes suchas RAD16, RAD3, RAD54 and SSL2 (see Table 2).Bang et al. (1995) observed transcriptional acti-vation of the RAD16 gene upon heat shock andupon UV irradiation. We also detected an increasein the level of transcripts of the same genes afterUV irradiation (see Table 2). In addition, tran-scriptional activation of 19 out of the 43 testedtranscripts (44·2%) was induced by UV irradiation;18 out of the 19 ORFs were also transcriptionallyactivated by heat shock. We do not know why therepair genes are transcriptionally activated uponheat shock and UV irradiation; however, thisphenonemon suggests that the same gene plays arole not only in response to UV damage, but alsoin response to other stress conditions. Geneswhich have similar expression profiles to both UVirradiation and heat shock are possibly involvedin the cellular response to both damages. Theyeast ORFs YGL064c, YGR271w, YKL078w,YLR247c, YLR276c, YHR169w, YKR024c,YMR290c and YNR038w may apply to this situ-ation. Furthermore, the specific role of heat-shock-responsible factors, such as Hsfp and Tsfp intranscriptional activation, remains unclear; how-ever, the nucleotide sequences of proteins whichbind these heat-shock activators, which are up to1 kb in length, were detected in the 5*-upstreamregion of 35 of 43 ORFs that were characterized(data not presented).

Induction of YER176w transcript during meiosisWe have also detected transcriptional activation

of YER176w during late meiosis (Table 2). Theprotein encoded by YER176w has been biochemi-cally identified as a DNA helicase I with unknownfunction (Bean and Matson, 1997). YER176w isallelic to the locus ecm32, which itself is related todefects in membrane function (Lussier et al., 1997).The protein encoded by YER176w may play a rolein DNA metabolism, such as in DNA repair orrecombination during the late stage of meiosis.

PerspectivesIn this study, we have considered the potential

importance of various cellular functions of

Yeast 15, 219–253 (1999)

248 . .

helicase-like proteins, and systematically identifiedand classified 134 yeast helicase-like ORFs in agenome-wide sequence analysis. Furthermore, 21previously uncharacterized unique ORFs werephenotypically characterized by gene disruption.Of the 21 previously uncharacterized ORFs, sevenwere found to be essential for vegetative growthand three loci were associated with the slow-growth phenotype. Furthermore, most of these 10genes have not been identified throughout theprevious genetic studies using various ts mutants.These suggest that the products of these helicase-related genes must play crucial and novel roles inthe transactions of DNA, RNA and chromatin inyeast cells, and that the systematic approachshown in this study is very useful for discoveringnew members of a particular gene family and theirnovel functions.

The specific biological functions of essentialyeast helicase-like proteins need to be clarified byseveral different approaches: more efficient expres-sion analyses by ‘DNA chip’ (DeRisi et al., 1997;Wodicka et al., 1997); exhaustive two-hybridscreening (Fromont-Racine et al., 1997); system-atic protein analysis by 2D-gel electrophoresis(Shevchenko et al., 1996); systematic creation andphenotypic analysis of conditional mutants; sev-eral kinds of biochemical assays using recombi-nant proteins, etc. An advantage to finding thebiological function of helicase-like genes exists,when compared with functional analyses of so-called ‘orphan genes’: the presence of helicasemotifs in helicase-like genes suggests that helicase-related proteins act on nucleic acids and chromatin.Furthermore, it has been known that helicase-likeproteins often interact with other proteins, andthat they function as a component of multiproteincomplexes such as the spliceosome, TFIIH, MCMcomplex, etc. (see Table 1). Indeed, Fromont-Racine et al. (1997) newly identified many helicase-related proteins that interact with authenticsplicing factors, and these proteins presumablyplay a role in the pre-mRNA splicing pathway.The protein linkage map of the helicase-relatedproteins identified in this study will facilitate theclarification of the cellular functions of these pro-teins, which are involved in a variety of cellularpathways. In addition, in the next several years, itwill become easier to identify homologues to yeasthelicase-like proteins in higher eukaryotes (S.pombe, C. elegans, Drosophila, mouse and human),by performing a homology search ‘in silico’ usingthe yeast protein sequence. Classification of


homologous sequences across species, and system-atic functional analysis of helicase-like genes, willgive us the opportunity to clarify the structuraland functional diversity of the helicase-like pro-teins and their evolution. Future studies will alsolead to the discovery of new helicase-like proteinsthat are involved in human clinical disorders.

ACKNOWLEDGEMENTS

We are indebted to Dr H. Uemura (NIBH, MITI)for the yeast strains, and Drs M. Kitada and H.Yang (Nippon Roche Research Center) and DrsH. Hagiwara, M. Naitou, Ms T. Mogi, K. Abe andE. Kohriki (RIKEN) for technical advice andsupport. This work was supported by grants fromthe Life Science Research Project of RIKEN, andthe Science and Technology Agency of Japan toT.E. and F.H., and by a Grant-in-Aid for Encour-agement of Young Scientists from the Ministry ofEducation, Science, Sports, and Culture of Japanto T.E.

REFERENCES

Aasland, R., Gibson, T. J. and Stewart, A. F. (1995).The PHD finger: implications for chromatin-mediatedtranscriptional regulation. Trend Biochem. Sci. 20,56–59.

Aasland, R., Stewart, A. F. and Gibson, T. (1996). TheSANT domain: a putative DNA-binding domain inthe SWI-SNF and ADA complexes, the transcrip-tional co-repressor N-CoR and TFIIIB. TrendsBiochem. Sci. 21, 87–88.

Altschul, S. F., Gish, W., Miller, W., Myers, E. W. andLipman, D. J. (1990). Basic local alignment searchtool. J. Mol. Biol. 215, 403–410.

Anderson, J. S. J. and Parker, R. P. (1998). The 3* to 5*degradation of yeast mRNA turnover that requiresthe SKI2 DEVH box protein and 3* to 5* exonucleasesof the exosome complex. EMBO J. 17, 1497–1506.

Arenas, J. E. and Abelson, J. N. (1997). Prp43: an RNAhelicase-like factor involved in spliceosome disas-sembly. Proc. Natl Acad. Sci. USA 94, 11,798–11,802.

Auble, D. T., Wang, D., Post, K. W. and Hahn, S.(1997). Molecular analysis of the SNF2/SWI2 proteinfamily member MOT1, an ATP-driven enzyme thatdissociates TATA-binding protein from DNA. Mol.Cell. Biol. 17, 4842–4851.

Bairoch, A. (1993). The PROSITE dictionary of sitesand patterns in proteins, its current status. Nucl. AcidsRes. 21, 3097–3103.

Baganz, F., Hayes, A., Marren, D., Gardner, D. C. J.and Oliver, S. G. (1997). Suitability of replacementmarkers for functional analysis studies in Saccharo-myces cerevisiae. Yeast 13, 1563–1573.

Yeast 15, 219–253 (1999)

249 -

Baker, T. A. and Bell, S. P. (1998). Polymerases andthe replisome: machines within machines. Cell 92,295–305.

Bang, D. D., Timmermans, V., Verhage, R., Zeeman,A. M., van de Putte, P. and Brouwer, J. (1995).Regulation of the Saccharomyces cerevisiae DNArepair gene RAD16. Nucl. Acids Res. 23, 1679–1685.

Barta, I. and Iggo, R. (1995). Autoregulation of ex-pression of the yeast Dbp2p ‘DEAD-box’ protein ismediated by sequences in the conserved DBP2 intron.EMBO J. 14, 3800–3808.

Baumeister, W., Walz, J., Zuhl, F. and Seemuller, E.(1998). The proteasome: paradigm of a self-compartmentalizing protease. Cell 92, 367–380.

Bean, D. W. and Matson, S. W. (1997). Identification ofthe gene encoding scHelI, a DNA helicase fromSaccharomyces cerevisiae. Yeast 13, 1465–1475.

Biswas, S. B., Chen, P. H. and Biswas, E. E. (1997).Purification and characterization of DNA polymeraseá-associated replication protein A-dependent yeastDNA helicase A. Biochemistry 36, 13,270–13,276.

Brandriss, M. C. and Falvey, D. A. (1992). Prolinebiosynthesis in Saccharomyces cerevisiae: analysis ofthe PRO3 gene, which encodes delta 1-pyrroline-5-carboxylate reductase. J. Bacteriol. 74, 3782–3788.[Erratum J. Bacteriol. 174, 5176.]

Budd, M. E. and Campbell, J. L. (1995). A yeast generequired for DNA replication encodes a protein withhomology to DNA helicases. Proc. Natl Acad. Sci.USA 92, 7642–7646.

Chang, R.-Y., Weaver, P. L., Liu, Z. and Chang, T.-H.(1997). Requirement of the DEAD-box proteinDed1p for messenger RNA translation. Science 275,1468–1471.

Chang, T. H., Arenas, J. and Abelson, J. (1990). Iden-tification of five putative yeast RNA helicase genes.Proc. Natl Acad. Sci. USA 87, 1571–1575.

Cherry, J. M., Adler, C., Ball, C., Chervitz, S. A.,Dwight, S. S., Hester, E. T., Jia, Y., Juvik, G., Roe,T., Schroeder, M., Weng, S. and Botstein, D. (1998).SGD: Saccharomyces Genome Database. Nucl. AcidsRes. 26, 73–79.

Chia, L. L. and McLaughlin, C. (1979). The half-life ofmRNA in Saccharomyces cerevisiae. Mol. Gen. Genet.170, 137–144.

Chong, J. P., Thommes, P. and Blow, J. J. (1996). Therole of MCM/P1 proteins in the licensing of DNAreplication. Trends Biochem. Sci. 21, 102–106.

Chua, P. and Roeder, G. S. (1995). Bdf1, a yeastchromosomal protein required for sporulation. Mol.Cell. Biol. 15, 3685–3696.

Clark, M. W., Zhong, W. W., Keng, T., Storms, R. K.,Barton, A., Kaback, D. B. and Bussey, H. (1992).Identification of a Saccharomyces cerevisiae homologof the SNF2 transcriptional regulator in the DNAsequence of an 8·6 kb region in the LTE1-CYS1interval of the left arm of chromosome I. Yeast 8,133–145.


Coissac, E., Maillier, E., Robineau, S. and Netter, P.(1996). Sequence of a 39,411 bp DNA fragmentcovering the left end of chromosome VII of Sac-charomyces cerevisiae. Yeast 12, 1555–1562.

Cole, G. M., Schild, D. and Mortimer, R. K. (1989).Two DNA repair and recombination genes in Sac-charomyces cerevisiae, RAD52 and RAD54, areinduced during meiosis. Mol. Cell. Biol. 9, 3101–3104.

Company, M., Arenas, J. and Abelson, J. (1991).Requirement of the RNA helicase-like protein PRP22for release of messenger RNA from spliceosomes.Nature 349, 487–493.

Confalonieri, F. and Duguet, M. (1995). A 200-aminoacid ATPase module in search of a basic function.Bioessays 17, 639–650.

Coster, F., Van Dyck, L., Jonniaux, J. L., Purnelle, B.and Goffeau, A. (1995). The sequence of a 13·5 kbDNA segment from the left arm of yeast chromo-some XIV reveals MER1; RAP1; a new putativemember of the DNA replication complex and a newputative serine/threonine phosphatase gene. Yeast 11,85–91.

Craig, E. A. (1992). The heat-shock response of Sac-charomyces cerevisiae. In Jones, E., Pringle, J. R. andBroach, J. R. (Eds), Molecular Biology of the YeastSaccharomyces: Gene Expression. Cold Spring HarborLaboratory Press, New York, pp. 501–537.

Cullmann, G., Fien, K., Kobayashi, R. and Stillman, B.(1995). Characterization of the five replication factorC genes of Saccharomyces cerevisiae. Mol. Cell Biol.15, 4661–4671.

Czaplinski, K., Weng, Y., Hagan, K. W. and Peltz,S. W. (1995). Purification and characterization of theUpf1 protein: a factor involved in translation andmRNA degradation. RNA 1, 610–623.

Daugeron, M. C. and Linder, P. (1998). Dbp7p, aputative ATP-dependent RNA helicase from Sac-charomyces cerevisiae, is required for 60S ribosomalsubunit assembly. RNA 4, 566–581.

Decottignies, A. and Goffeau, A. (1997). Completeinventory of the yeast ABC proteins. Nature Genet.15, 137–145.

DeRisi, J. L., Iyer, V. R. and Brown, P. O. (1997).Exploring the metabolic and genetic control of geneexpression on a genome scale. Science 278, 680–686.

Dhawale, S. S. and Lane, A. C. (1993). Compilation ofsequence-specific DNA binding proteins implicated intranscriptional control in fungi. Nucl. Acids Res. 21,5537–5546.

DiDomenico, B. J., Bugaisky, G. E. and Lindquist, S.(1982). Heat shock and recovery are mediated bydifferent translational mechanisms. Proc. Natl AcadSci. USA 79, 6181–6185.

Eisen, J. A., Sweder, K. S. and Hanawalt, P. C. (1995).Evolution of the SNF2 family of proteins: subfamilieswith distinct sequences and functions. Nucl. AcidsRes. 23, 2715–2723.

Yeast 15, 219–253 (1999)

250 . .

Eki, T., Naitou, M., Hagiwara, H., Abe, M. et al.(1996). Fifteen open reading frames in a 30·8 kbregion of the right arm of chromosome VI fromSaccharomyces cerevisiae. Yeast 12, 177–190.

Elledge, S. J., Zhou, Z., Allen, J. B. and Navas, T. A.(1993). DNA damage and cell cycle regulation ofribonucleotide reductase. Bioessays 15, 333–339.

Elledge, S. J. (1996). Cell cycle checkpoints: preventingan identity crisis. Science 274, 1664–1672.

Ellis, N. A., Gorden, J., Ye, T.-Z., Straughen, J., Ciocci,S., Lennon, D. J., Proytcheva, M., Alhadeff, B. andGerman, J. (1995). The Bloom’s syndrome geneproduct is homologous to RecQ helicases. Cell 83,655–666.

Ellis, N. A. (1997). DNA helicases in inherited humandisorders. Curr. Opin. Genet. Dev. 7, 354–363.

Fleckner, J., Zhang, M., Valcarcel, J. and Green, M. R.(1997). U2AF65 recruits a novel human DEAD boxprotein required for the U2 snRNP-branchpointinteraction. Genes Dev. 11, 1864–1872.

Fraser, R. A., Rossignol, M., Heard, D. J., Egly, J. M.and Chambon, P. (1997). SUG1, a putative transcrip-tional mediator and subunit of the PA700 proteasomeregulatory complex, is a DNA helicase. J. Biol. Chem.272, 7122–7126.

Friedberg, E. C., Siede, W. and Cooper, A. J. (1991).Cellular responses to DNA damage in yeast. In Jones,E., Pringle, J. R. and Broach, J. R. (Eds), MolecularBiology of the Yeast Saccharomyces: GenomeDynamics, Protein Synthesis and Energetics. ColdSpring Harbor Laboratory Press, New York,pp. 147–192.

Fromont-Racine, M., Rain, J.-C. and Legrain, P. (1997).Toward a functional analysis of the yeast genomethrough exhaustive two-hybrid screens. Nature Genet.16, 277–282.

Gerring, S. L., Spencer, F. and Hieter, P. (1990). TheCHL 1 (CTF 1) gene product of Saccharomycescerevisiae is important for chromosome transmissionand normal cell cycle progression in G2/M. EMBO J9, 4347–4358.

Gibbons, R. J., Picketts, D. J., Villard, L. and Higgs,D. R. (1995). Mutations in a putative global transcrip-tional regulator cause X-linked mental retardationwith á-thalassemia (ATR-X syndrome). Cell 80,837–845.

Gietz, R. D., Schiestl, R. H., Willems, A. R. and Woods,R. A. (1995). Studies on the transformation of intactyeast cells by LiAc/SS-DNA/PEG procedure. Yeast11, 355–360.

Goffeau, A. et al.1997). The yeast genome directory.Nature 387 (Suppl.), 5–105.

Gorbalenya, A. E. and Koonin, E. V. (1993). Helicases:amino acid sequence comparisons and structure–function relationships. Curr. Biol. 3, 419–429.

Grant, P. A., Duggan, L., Cote, J., Roberts, S. M.,Brownell, J. E., Candau, R., Ohba, R., Owen-Hughes,T., Allis, C. D., Winston, F., Berger, S. L. and


Workman, J. L. (1997). Yeast Gcn5 functions in twomultisubunit complexes to acetylate nucleosomalhistones: characterization of an Ada complex and theSAGA (Spt/Ada) complex. Genes Dev. 11, 1640–1650.

Guzder, S. N., Sung, P., Bailly, V., Prakash, L. andPrakash, S. (1994). RAD25 is a DNA helicaserequired for DNA repair and RNA polymerase IItranscription. Nature 369, 578–581.

Guzder, S. N., Habraken, Y., Sung, P., Prakash, L. andPrakash, S. (1996). RAD26, the yeast homolog ofhuman Cockayne’s syndrome group B gene, encodesa DNA-dependent ATPase. J. Biol. Chem. 271,18,314–18,317.

Hampsey, M. (1997). A review of phenotypes in Sac-charomyces cerevisiae. Yeast 13, 1099–1133.

Henikoff, S., Greene, E. A., Pietrokovski, S., Bork, P.,Attwood, T. K. and Hood, L. (1997). Gene families:the taxonomy of protein paralogs and chimeras.Science 278, 609–614.

Hodges, P. E., Payne, W. E. and Garrels, J. I. (1998).The Yeast Protein Database: a curated proteomedatabase for Saccharomyces cerevisiae. Nucl. AcidsRes. 26, 68–72.

Hofmann, K. and Stoffel, W. (1993). TMBASE—Adatabase of membrane spanning protein segments.Biol. Chem. Hoppe-Seyler 374, 166.

Hunter, T. and Plowman, G. D. (1997). The proteinkinases of budding yeast: six score and more. Trend.Biochem. Sci. 22, 18–22.

Ishimi, Y. (1997). A DNA helicase activity is associatedwith an MCM4, -6, and -7 protein complex. J. Biol.Chem. 272, 24,508–24,513.

Jacobson, A. and Peltz, S. W. (1996). Interrelationshipsof the pathways of mRNA decay and translation ineukaryotic cells. Ann. Rev. Biochem. 65, 693–739.

James, C. M., Indge, K. J. and Oliver, S. G. (1995).DNA sequence analysis of a 35 kb segment fromSaccharomyces cerevisiae chromosome VII reveals 19open reading frames including RAD54, ACE/CUP2,PMR1, RCK1, AMS1 and CAL1/CDC43. Yeast 11,1413–1419.

Jamieson, D. J. and Beggs, J. D. (1991). A suppressor ofyeast spp81/ded1 mutations encodes a very similarputative ATP-dependent RNA helicase. Mol. Micro-biol. 5, 805–812.

Jeanmougin, F., Wurtz, J.-M., Le Douarin, B.,Chanbon, P. and Losson, R. (1997). The bromo-domain revisited. Trends Biochem. Sci. 22, 151–153.

Johnson, R. E., Prakash, S. and Prakash, L. (1994).Yeast DNA repair protein RAD5 that promotesinstability of simple repetitive sequences is a DNA-dependent ATPase. J. Biol. Chem. 269, 28,259–28,262.

Kadonaga, J. T. (1998). Eukaryotic transcription: aninterlaced network of transcription factors andchromatin-modifying machines. Cell 92, 307–313.

Kaiser, C., Michaelis, S. and Mitchell, A. (Eds) (1994).Methods in Yeast Genetics, 1994 edn. Cold SpringHarbor Press, New York.

Yeast 15, 219–253 (1999)

251 -

Karow, J. K., Chakraverty, R. K. and Hickson, I. D.(1997). The Bloom’s syndrome gene product is a 3*–5*DNA helicase. J. Biol. Chem. 272, 30,611–30,614.

Kim, S. H., Smith, J., Claude, A. and Lin, R. J. (1992).The purified yeast pre-mRNA splicing factor PRP2is an RNA-dependent NTPase. EMBO J. 11,2319–2326.

Kitada, K., Yamaguchi, E. and Arisawa, M. (1995).Cloning of the Candida glabrata TRP1 and HIS3genes, and construction of their disruptant strainsby sequential integrative transformation. Gene 165,203–206.

Klein, H. L. (1997). RDH54, a RAD54 homologue inSaccharomyces cerevisiae, is required for mitoticdiploid-specific recombination and repair and formeiosis. Genetics 147, 1533–1543.

Kogoma, T., Cadwell, G. W., Barnard, K. G. and Asai,T. (1996). The DNA replication priming protein,PriA, is required for homologous recombinationand double-strand break repair. J. Bacteriol. 178,1258–1264.

Koonin, E. V. (1992). A new group of putative RNAhelicases. Trend. Biochem. Sci. 17, 495–497.

Koonin, E. V. (1993a). A common set of conservedmotifs in a vast variety of putative nucleic acid-dependent ATPases including MCM proteinsinvolved in the initiation of eukaryotic DNAreplication. Nucl. Acids Res. 21, 2541–2547.

Koonin, E. V. (1993b). A superfamily of ATPases withdiverse functions containing either classical or deviantATP-binding motif. J. Mol. Biol. 229, 1165–1174.

Koonin, E. V. and Rudd, K. E. (1996). Two domains ofsuperfamily I helicases may exist as separate proteins.Protein Science 5, 178–180.

Kouprina, N., Kroll, E., Kirillov, A., Bannikov, V.,Zakharyev, V. and Larionov, V. (1994). CHL12, agene essential for the fidelity of chromosome transmis-sion in the yeast Saccharomyces cerevisiae. Genetics138, 1067–1079.

Kressler, D., de la Cruz, J., Rojo, M. and Linder, P.(1997). Fal1p is an essential DEAD-box proteininvolved in 40S-ribosomal-subunit biogenesis inSaccharomyces cerevisiae. Mol. Cell. Biol. 17,7283–7294.

Kressler, D., de la Cruz, J., Rojo, M. and Linder, P.(1998). Dbp6p is an essential putative ATP-dependentRNA helicase required for 60S-ribosomal-subunitassembly in Saccharomyces cerevisiae. Mol. Cell. Biol.18, 1855–1865.

Lahaye, A., Stahl, H., Thines-Sempoux, D. and Foury,F. (1991). PIF1: a DNA helicase in yeast mitochon-dria. EMBO J 10, 997–1007.

Laurent, B. C., Yang, X. and Carlson, M. (1992). Anessential Saccharomyces cerevisiae gene homologousto SNF2 encodes a helicase-related protein in a newfamily. Mol. Cell. Biol. 12, 1893–1902.

Laurent, B. C., Treich, I. and Carlson, M. (1993).The yeast SNF2/SWI2 protein has DNA-stimulated


ATPase activity required for transcriptionalactivation. Genes Dev. 7, 583–591.

Lee, C. G., Chang, K. A., Kuroda, M. I. and Hurwitz, J.(1997). The NTPase/helicase activities of Drosophilamaleless, an essential factor in dosage compensation.EMBO J. 16, 2671–2681.

Li, X. and Burgers, P. M. (1994). Molecular cloning andexpression of the Saccharomyces cerevisiae RFC3gene, an essential component of replication factor C.Proc. Natl Acad. Sci. USA 91, 868–872.

Liang, S., Hitomi, M., Hu, Y. H., Liu, Y. and Tartakoff,A. M. (1996). A DEAD-box-family protein is requiredfor nucleocytoplasmic transport of yeast mRNA.Mol. Cell. Biol. 16, 5139–5146.

Liang, W. Q., Clark, J. A. and Fournier, M. J. (1997).The rRNA-processing function of the yeast U14 smallnucleolar RNA can be rescued by a conserved RNAhelicase-like protein. Mol. Cell. Biol. 17, 4124–4132.

Linder, P. and Slonimski, P. P. (1989). An essential yeastprotein, encoded by duplicated genes TIF1 and TIF2and homologous to the mammalian translation in-itiation factor eIF-4A, can suppress a mitochondrialmissense mutation. Proc. Natl Acad. Sci. USA 86,2286–2290.

Lindquist, S. (1981). Regulation of protein synthesisduring heat shock. Nature 293, 311–314.

Lohman, T. M. and Bjornson, K. P. (1996). Mech-anisms of helicase-catalyzed DNA unwinding. Ann.Rev. Biochem. 65, 169–214.

Louis, E. J. (1995). The chromosome ends of Saccharo-myces cerevisiae. Yeast 11, 1553–1573.

Lu, J., Mullen, J. R., Brill, S. J., Kleff, S., Romeo, A. M.and Sternglanz, R. (1996a). Human homologues ofyeast helicase. Nature 383, 678–679.

Lu, J., Kobayashi, R. and Brill, S. J. (1996b). Charac-terization of a high mobility group 1/2 homolog inyeast. J. Biol. Chem. 271, 33,678–33,685.

Lussier, M., White, A.-M., Sheraton, J., di Paolo, T.,Treadwell, J. et al. (1997). Large scale identification ofgenes involved in cell surface biogenesis and architec-ture in Saccharomyces cerevisiae. Genetics 147,435–450.

Makino, Y., Yamano, K., Kanemaki, M., Morikawa,K., Kishimoto, T., Shimabara, N., Tanaka, K. andTamura, T. (1997). SUG1, a component of the 26Sproteasome, is an ATPase stimulated by specificRNAs. J. Biol. Chem. 272, 23,201–23,205.

Marians, K. J. (1997). Helicase structures: a new twiston DNA unwinding. Structure 5, 1129–1134.

Martegani, E., Vanoni, M., Mauri, I., Rudoni, S.,Saliola, M. and Alberghina, L. (1997). Identificationof a gene encoding a putative RNA-helicase, homolo-gous to SKI2, in chromosome VII of Saccharomycescerevisiae. Yeast 13, 391–397.

Matson, S. W. and Kaiser-Rogers, K. A. (1990). DNAhelicases. Ann. Rev. Biochem. 59, 289–329.

Mewes, H. W., Albermann, K., Bahr, M., Frishman, D.,Gleissner, A., Hani, J., Heumann, K., Kleine, K.,

Yeast 15, 219–253 (1999)

252 . .

Maierl, A., Oliver, S. G., Pfeiffer, F. and Zollner, A.(1997). Overview of the yeast genome. Nature 387(Suppl.), 7–65.

Mewes, H. W., Hani, J., Pfeiffer, F. and Frishman, D.(1998). MIPS: a database for protein sequences andcomplete genomes. Nucl. Acids Res. 26, 33–37.

Mitchell, P. A. (1994). Control of meiotic gene ex-pression in Saccharomyces cerevisiae. Microbiol. Rev.58, 56–70.

Naitou, M., Hagiwara, H., Hanaoka, F., Eki, T. andMurakami, Y. (1997). Expression profiles of tran-scripts from 126 open reading frames in the entirechromosome VI of Saccharomyces cerevisiae bysystematic Northern analyses. Yeast 13, 1275–1290.

Nakai, K. and Kanehisa, M. (1992). A knowledge basefor predicting protein localization sites in eukaryoticcells. Genomics 14, 897–911.

Noble, S. M. and Guthrie, C. (1996). Identification ofnovel genes required for yeast pre-mRNA splicingby means of cold-sensitive mutations. Genetics 143,67–80.

O’Day, C. L., Chavanikamannil, F. and Abelson, J.(1996a). 18S rRNA processing requires the RNAhelicase-like protein Rrp3. Nucl. Acids Res. 24,3201–3207.

O’Day, C. L., Dalbadie-McFarland, G. and Abelson, J.(1996b). The Saccharomyces cerevisiae Prp5 proteinhas RNA-dependent ATPase activity with specificityfor U2 small nuclear RNA. J. Biol. Chem. 271,33,261–33,267.

Page, R. D. M. (1996). TREEVIEW: an application todisplay phylogenetic trees on personal computers.Comp. Appl. Biosci. 12, 357–358.

Petukhova, G., Stratton, S. and Sung, P. (1998). Cata-lysis of homologus DNA pairing by yeast Rad51 andRad54 proteins. Nature 393, 91–94.

Razin, M. J. and Kadonaga, J. T. (1997). SWI2/SNF2and related proteins: ATP-driven motors that disruptprotein-DNA interactions? Cell 88, 737–740.

Rieger, K.-J., Kaniak, A., Cooppee, J.-Y., Alijinovic,G., Baudin-Baillieu, A., Orlowska, R., Groudinsky,O., Di Rago, J.-P. and Slonimski, P. P. (1997).Large-scale phenotypic analysis—the pilot project onyeast chromosome III. Yeast 13, 1547–1562.

Ripmaster, T. L., Vaughn, G. P. and Woolford, J. L., Jr(1992). A putative ATP-dependent RNA helicaseinvolved in Saccharomyces cerevisiae ribosome assem-bly. Proc. Natl Acad. Sci. USA 89, 11,131–11,135.

Robbins, J., Dilworth, S. M., Laskey, R. A. andDingwall, C. (1991). Two interdependent basicdomains in nucleoplasmin nuclear targeting sequence:identification of a class of bipartite nuclear targetingsequence. Cell 64, 615–623.

Rong, L. and Klein, H. L. (1993). Purification andcharacterization of the SRS2 DNA helicase of theyeast Saccharomyces cerevisiae. J. Biol. Chem. 268,1252–1259.


Raymond, B. C. and Rosbash, M. (1992). Yeast pre-mRNA splicing. In Jones, E., Pringle, J. R. andBroach, J. R. (Eds), Molecular Biology of the YeastSaccharomyces: Gene Expression. Cold Spring HarborLaboratory Press, New York, pp. 143–192.

Sachs, A. B. and Davis, R. W. (1990). Translationinitiation and ribosomal biogenesis: involvement of aputative rRNA helicase and RPL46. Science 247,1077–1079.

Saren, A. M., Laamanen, P., Lejarcegui, J. B. andPaulin, L. (1997). The sequence of a 36·7 kb segmenton the left arm of chromosome IV from Saccharo-myces cerevisiae reveals 20 non-overlapping openreading frames (ORFs) including SIT4, FAD1,NAM1, RNA11, SIR2, NAT1, PRP9, ACT2 andMPS1 and 11 new ORFs. Yeast 13, 65–71.

Schild, D., Glassner, B. J., Mortimer, R. K., Carlson,M. and Laurent, B. C. (1992). Identification ofRAD16, a yeast excision repair gene homologous tothe recombinational repair gene RAD54 and to theSNF2 gene involved in transcriptional activation.Yeast 8, 385–395.

Schmid, S. R. and Linder, P. (1991). Translationinitiation factor 4A from Saccharomyces cerevisiae:analysis of residues conserved in the D-E-A-Dfamily of RNA helicases. Mol. Cell. Biol. 11,3463–3471.

Schmid, S. R. and Linder, P. (1992). D-E-A-D proteinfamily of putative RNA helicases. Mol. Microbiol. 6,283–291.

Schnall, R., Mannhaupt, G., Stucka, R., Tauer, R.,Ehnle, S., Schwarzlose, C., Vetter, I. and Feldman, H.(1994). Identification of a set of yeast genes coding fora novel family of putative ATPases with high simi-larity to constituents of the 26S proteasome complex.Yeast 10, 1141–1155.

Schwer, B. and Guthrie, C. (1991). PRP16 is an RNA-dependent ATPase that interacts transiently with thespliceosome. Nature 349, 494–499.

Schwer, B. and Gross, C. H. (1998). Prp22, a DExH-boxRNA helicase, plays two distinct roles in yeastpre-mRNA splicing. EMBO J. 17, 2086–2094.

Seraphin, B., Simon, M., Boulet, A. and Faye, G.(1989). Mitochondrial splicing requires a protein froma novel helicase family. Nature 337, 84–87.

Shevchenko, A., Jensen, O. N., Podtelejnikov, A. V.,Sagiocco, F., Wilm, M., Vorm, O., Mortensen, P.,Shevchenko, A., Boucherie, H. and Mann, M. (1996).Linking genome and proteome by massspectrometry—large scale identification of yeast pro-teins from 2-dimensional gels. Proc. Natl Acad. Sci.USA 93, 14,440–14,445.

Sinclair, D. A. and Guarante, L. (1997). Extrachromo-somal rDNA circles—a cause of aging in yeast. Cell91, 1033–1042.

Sinclair, D. A., Mills, K. and Guarente, L. (1997).Accelerated aging and nucleolar fragmentation inyeast sgs1 mutants. Science 277, 1313–1316.

Yeast 15, 219–253 (1999)

253 -

Staley, J. P. and Guthrie, C. (1998). Mechanical devicesof the spliceosome: motors, clocks, springs, andthings. Cell 92, 315–326.

Steinmetz, E. J. and Brow, D. A. (1996). Repression ofgene expression by an exogenous sequence elementacting in concert with a heterogenous nuclearribonucleoprotein-like protein, Nrd1, and the putativehelicase Sen1. Mol. Cell. Biol. 16, 6993–7003.

Stepien, P. P., Margossian, S. P., Landsman, D. andButow, R. A. (1992). The yeast nuclear gene suv3affecting mitochondrial post-transcriptional processesencodes a putative ATP-dependent RNA helicase.Proc. Natl Acad. Sci. USA 89, 6813–6817.

Strahl-Bolsinger, S. and Tanner, W. (1993). A yeast geneencoding a putative RNA helicase of the ‘‘DEAD’’-box family. Yeast 9, 429–432.

Strauss, E. J. and Guthrie, C. (1994). PRP28, a ‘DEAD-box’ protein, is required for the first step of mRNAsplicing in vitro. Nucl. Acids Res. 22, 3187–3193.

Sun, Z. W. and Hampsey, M. (1996). Synthetic enhance-ment of a TFIIB defect by a mutation in SSU72, anessential yeast gene encoding a novel protein thataffects transcription start site selection in vivo. Mol.Cell. Biol. 16, 1557–1566.

Sung, P., Prakash, L., Matson, S. W. and Prakash, S.(1987). RAD3 protein of Saccharomyces cerevisiae isa DNA helicase. Proc. Natl Acad. Sci. USA 84,8951–8955.

Suzuki, N., Shimamoto, A., Imamura, O., Kuromitsu,J., Kitao, S., Goto, M. and Furuichi, Y. (1997). DNAhelicase activity in Werner’s syndrome gene productsynthesized in a baculovirus system. Nucl. Acids Res.25, 2973–2987.

Takeuchi, K., Kawashima, A., Nagafuchi, A. andTsukita, S. (1994). Structural diversity of band 4.1superfamily members. J. Cell Sci. 107, 1921–1928.

Tanaka, K., Matsumoto, K. and Toh-e, A. (1988). Dualregulation of the expression of the polyubiquitin geneby cyclic AMP and heat shock in yeast. EMBO J. 7,495–502.

Thompson, J. D., Higgins, D. G. and Gibson, T. J.(1994). CLUSTAL W: improving the sensitivity ofprogressive multiple sequence alignment throughsequence weighting, positions-specific gap penaltiesand weight matrix choice. Nucl. Acids Res. 22,4673–4680.

Tseng, S. S.-I., Weaver, P. L., Liu, Y., Hitomi, M.,Tartakoff, A. M. and Chang, T.-H. (1998). Dbp5p, acytosolic RNA helicase, is required for poly(A)+ RNAexport. EMBO J. 17, 2651–2662.


Velculescu, V. E., Zhang, L., Zhou, W., Vogelstein, J.,Basrai, M. A., Bassett, D. E., Jr, Hieter, P.,Vogelstein, B. and Kinzler, K. W. (1997). Charac-terization of the yeast transcriptome. Cell 88,243–251.

Venema, J., Bousquet-Antonelli, C., Gelugne, J. P.,Caizergues-Ferrer, M. and Tollervey, D. (1997).Rok1p is a putative RNA helicase required for rRNAprocessing. Mol. Cell. Biol. 17, 3398–3407.

Wang, Y., Wagner, J. D. O. and Guthrie, C. (1998). TheDEAH-box splicing factor Prp16 unwinds RNAduplexes in vitro. Curr. Biol. 8, 441–451.

Weaver, P. L., Sun, C. and Chang, T. H. (1997). Dbp3p,a putative RNA helicase in Saccharomyces cerevisiae,is required for efficient pre-rRNA processing pre-dominantly at site A3. Mol. Cell. Biol. 17, 1354–1365.

Weeda, G., Rossignol, M., Fraser, R. A., Winkler,G. S., Vermeulen, W., van’t Veer, L. J., Ma, L.,Hoeijmakers, J. H. J. and Egly, J.-M. (1997). TheXPB subunit of repair/transcription factor TFIIHdirectly interacts with SUG1, a subunit of the 26Sproteasome and putative transcription factor. Nucl.Acids Res. 25, 2274–2283.

Wickner, R. B. (1996). Double-stranded RNA virusesof Saccharomyces cerevisiae. Microbiol. Rev. 60,250–265.

Wodicka, L., Dong, H., Mittmann, M., Ho, M.-H. andLockhart, D. L. (1997). Genome-wide expressionmonitoring in Saccharomyces cerevisiae. NatureBiotech. 15, 1359–1367.

Wolfe, K. H. and Shields, D. C. (1997). Molecularevidence for an ancient duplication of the entire yeastgenome. Nature 387, 708–713.

Woodage, T., Basrai, M. A., Baxenvanis, A. D., Hieter,P. and Collins, F. S. (1997). Characterization of theCHD family of proteins. Proc. Natl Acad. Sci. USA94, 11,471–11,477.

Worley, K. C., Wiese, B. A. and Smith, R. F. (1995).BEAUTY: an enhanced BLAST-based search toolthat integrates multiple biological informationresources into sequence similarity search results.Genome Res. 5, 173–184.

Yu, C.-E., Oshima, J., Fu, Y.-H., Wijsman, E. M.,Hisama, F., Alisch, R., Matthews, S., Nakura, J.,Miki, T. et al. (1996). Positional cloning of theWerner’s syndrome gene. Science 272, 258–262.

Zhang, Z. and Buchman, A. R. (1997). Identification ofa member of a DNA-dependent ATPase family thatcauses interference with silencing. Mol. Cell. Biol. 17,5461–5472.

Yeast 15, 219–253 (1999)

Documents

Systematic identification, classification, and characterization of the open reading frames which encode novel helicase-related proteins inSaccharomyces cerevisiae by gene disruption