Multiple Pathways for Homologous Recombination in ...46 A. J. Rattray and L. S. Symington that are required for recombination in Saccharomyces cerevisiae, it has not been clearly demonstrated

Copyright 0 1995 by the Genetics Society of America

Multiple Pathways for Homologous Recombination in Saccharomyces cereuiSiae

Alison J. Rattray and Lorraine S. Symington

Department of Microbiology and Institute for Cancer Research, Columbia University College of Physicians and Surgeons, New York, New York 10032

Manuscript received July 11, 1994 Accepted for publication September 12, 1994

ABSTRACT The genes in the RAD52 epistasis group of Saccharomyces cevevisiae are necessary for most mitotic

and meiotic recombination events. Using an intrachromosomal inverted-repeat assay, we previously demonstrated that mitotic recombination of this substrate is dependent upon the RAD52 gene. In the present study the requirement for other genes in this epistasis group for recombination of inverted repeats has been analyzed, and double and triple mutant strains were examined for their epistatic relationships. The majority of recombination events are mediated by a RAD51-dependent pathway, where the RAD54, RAD55 and RAD57genes function downstream of M51. Cells mutated in RAD55 or RAD57 as well as double mutants are cold-sensitive for inverted-repeat recombination, whereas a rad51 rad55 rad57 triple mutant is not. The RAD1 gene is not required for inverted-repeat recombination but is able to process spontaneous DNA lesions to produce recombinant products in the absence of RAD51. Furthermore, there is still considerably more recombination in rad1 rad51 mutants than in rad52mutants, indicating the presence of another, as yet unidentified, recombination pathway.

S TUDIES of meiotic recombination in ascomycete fungi, where all of the products of a recombination

event can be recovered, have shown a strong association between gene conversion and crossing over (see PETES et al. 1991 for a review). These observations have been accounted for in models for recombination in which heteroduplex DNA is proposed as an intermediate in the formation of crossover products (HOLLIDAY 1964; MESELSON and RADDINC 1975; RADDINC 1982; SZOSTAK et al. 1983). The models predict that gene conversion, the result of heteroduplex repair, is associated with crossing over 50% of the time. However, the isolation of mutations that differentially affect conversion and crossing over in meiosis (ENGEBRECHT et al. 1990; ROCK- MILL and ROEDER 1990) suggests that there might be separate pathways for each type of event. The concur- rence of gene conversions and crossovers is difficult to establish in mitotic cells because usually only one product of a recombination event is recovered. Instead, it is generally inferred from the relative proportions of each type of event among the total recombinant progeny. ROMAN and RUZINSKI (1990) found evidence that the majority of gene conversion events occur in the G1 phase of the cell cycle, whereas the majority of crossovers occurred during G2. This temporal separation is suggestive of separate pathways for gene conversions and crossovers. Although all crossovers may require a prior gene conversion event (or heteroduplex precur- sor), there is evidence that shorter gene conversion tracts are preferentially noncrossover, and longer con-

Cmesponding author: Lorraine S. Symington, Institute of Cancer Research, Columbia University College of Physicians and Surgeons, 701 W. 168th St., Room 912, New York, NY 10032.

Genetics 139: 45-56 (Janualy, 1995)

version tracts show a greater association with crossovers (AHN and LMNCSTON 1986; AGUILERA and KLEIN 1989). Therefore, there may be a different set or subset of gene products that dictate preferentially noncrossover recombination products in G1.

Although recombination has been well characterized in Escherichia coli, few studies have examined the association between gene conversion and crossing over because in most assays both products of a recombination event are not recovered. To examine the issue of recip- rocality of recombination events, SECALL and R o T H (1994) developed a chromosomal inverted-repeated system in Salmonella typhimunum. In this system four classes of recombinant products were recovered: inversions, inversions associated with conversion, apparent gene conversions and double crossovers. In recA mutants all classes of recombination events were reduced by lo4- fold. In recB mutants the inversion class was eliminated, and the other classes reduced 2- to 60-fold. These data provide evidence that gene conversions and crossovers can proceed through different pathways in bacteria.

The concept of recombination pathways was developed from the genetic analysis of conjugative recombination in E. coli (reviewed in SMITH 1989). The RecBCD pathway is the primary pathway for conjugative recombination; the RecE and RecF pathways are activated by suppressor mutations in the absence of recB or recC function. All of the recombination pathways, RecBCD, RecE and RecF, require the recA gene product, and most of the genes required for the RecE pathway, with the exception of re&, are required for the RecF pathway. Thus there is considerable overlap between these pathways. Although several genes have been identified

46 A. J. Rattray and L. S. Symington

that are required for recombination in Saccharomyces cerevisiae, it has not been clearly demonstrated whether these function in a common pathway. The major group of genes involved in mitotic recombination is the RAD52 epistasis group (GAME 1983, 1993). These genes are characterized by their requirement for repair of ionizing radiation-induced DNA damage (GAME and MORTIMER 1974). Studies of mitotic recombination between directly repeated sequences have identified two alternate pathways that require the W 5 2 and RAD1 gene products, respectively, indicating that alternate pathways for recombination exist in yeast (KLEIN 1988; SCHIESTL and PRAKASH 1988; THOMAS and ROTHSTEIN 1989; ZEHFUS et al. 1990). The RAD1 gene is required for excision repair of UV-induced DNA damage, but mutants have no other known defects in mitotic or meiotic recombination.

We recently developed a chromosomal inverted-repeat assay for studying mitotic recombination in haploid yeast cells. Three classes of recombination products were recovered: gene conversions, crossovers (inversions) and crossovers associated with gene conversion (Figure 1). In rad52 mutants all classes of events were greatly reduced (>5000-fold), whereas a rad51 mutation reduced gene conversion events 18-fold and crossovers (both with or without an associated conversion) only 2.5-fold (RATTRAY and SYMINGTON 1994). Analysis of recombination in these two mutants was of particular interest because recent studies have shown a physical association between the W 5 1 and RAD52 gene products (SHINOHARA et al. 1992; MILNE and WEAVER 1993) and because Rad51 has substantial homology to the E. coli RecA protein (ABOUSSEKHRA et al. 1992; BASILE et al. 1992; SHINOHARA et al. 1992). The four-fold reduction in the rate of recombination between inverted repeats in rad51 mutants sharply contrasts with the lo4- fold reduction observed in S. typhimurium recA mutants. Also the different phenotypes of rad51 and rad52 mutants in this assay suggest that Rad52 has other functions in addition to interaction with Rad51.

In the current study this genetic analysis has been extended to include other members of the RAD52 epistasis group, as well as rad1 mutants. In addition, double and triple mutant strains have been examined to determine their epistatic relationships. Our data provide evidence for the existence of at least three distinct RAD52- dependent recombination pathways in yeast. The major pathway requires the RAD51, RAD54, RAD55 and RAD57 genes, a MI-dependent pathway and at least one other pathway. Furthermore, we show that mutation of genes in the RRD51-dependent pathway results in a significant decrease in the class of events corresponding to gene conversion events unassociated with crossing over.

MATERIALS AND METHODS Media, growth conditions and genetic methods: Rich me-

dium (YEPD), synthetic complete (SC) medium lacking the

appropriate amino acid or nucleic acid base, and 5-fluorooro- tic acid (5-FOA) medium were prepared as described (SHER- MAN et al. 1986). Cells were grown at 30" unless othemise indicated. Transformations were performed by the LAC method (IT0 et ai. 1983) and tetrad dissection as described previously (SHERMAN et al. 1986). The y-radiation sensitivity of strains was scored by replica plating test strains to YEPD medium and irradiating with 50 krad from a Gammacell-220 irradiator containing "Co (Atomic Energy of Canada, Ottawa, Canada). The UV sensitivity of strains was scored by replica plating test strains to YEPD medium and irradiating with 50 J/m' at 254 nm. For survival curves, cells were grown in liquid YEPD medium to early log phase and dilutions of cells were plated onto solid YEPD and irradiated at various doses.

Plasmids: A list of plasmids and their relevant sources is shown in Table 1. Construction of the plasmid used for inte- grating the inverted-repeat substrate has been described previously (RATTRAY and SYMINGTON 1994). Briefly, one repeat (ade2-n) consists of a 3.1-kilobase (kb) BgtII-SpeI fragment of the wild-type ADE2 gene that has been mutagenized by fill-in of the unique NdeI site at codon 465 with the Klenow fragment of DNA polymerase I. The second repeat (ade2-5'n) is a 1.8- kb EcoRV fragment of ADE2 that lacks the promoter and the first 225 bp of the ADE2 open reading frame. The two copies are separated by a 0.85-kb fragment containing the TRPl gene.

Yeast strains: A list of yeast strains and their relevant sources is shown in Table 2. All strains are derivatives of strains W3031A or W303-1B (THOMAS and ROTHSTEIN 1989). Strains yAR71 and yAR91 contain the nde2-SA-TRPlade2-n construct at the native HIS3 locus (Figure ZA). Strains yAR82, yAR86, yAR107, yARlO8 and yARl39 were constructed by one-step transplacement (ROTHSTEIN 1983) of yAR71 or ym91 (as indicated in Table 2) with the appropriate plasmid DNA fragments to generate disruption alleles of the indicated RAD genes. To construct strain yAR167 the URA3 gene was first excised from ade2::hisGURA3-hisG of strain yAR160 by selec- tion on 5-FOA medium (WINSTON et al. 1983) and then transformed to Ura+ with an EcoRI fragment from pNKY83. Strains yAR188 and yARl90 were constructed by transformation of yAR97 to Leu+ with an Xbal-PstI fragment of pAM28 or a Hind111 fragment of pL962, respectively. All other strains were constructed by mating appropriate strains (as indicated in Table 2) and selecting for haploid spores segregating the appropriate rad mutation(s), the ade2-5'A-TRPI-ade2-n inverted-repeat substrate and the ade2::URM disruption at the native ADE2 locus. The presence of the rad mutations was monitored by sensitivity to ionizing radiation (or UV irradiation for radl), and all strain constructions were further veri- fied by Southern blot analysis. We noted that a diploid heterc- zygous for rad51 rad55 and rad57mutations sporulated poorly at 23" (2-3%) and somewhat more efficiently at 30" (30- 40%), although the asci were extremely fragile, requiring no glusulase pretreatment before dissection. Single and double heterozygotes for any of these mutations sporulated with wild- type efficiency (>70%).

Determination of mitotic recombmation frequencies: At least two independent isolates of each strain were used for the determination of recombination rates. Single colonies Of

each isolate were grown on YEPD for 2-3 days. Growth for longer than 4 days gave artificially high recombination rates because Ade+ cells continue to divide for longer than Ade- cells. At least 27 pink colonies of each strain (usually 9 from each independent isolate) were resuspended in water and plated at the appropriate dilutions to determine total cell number (SC-Trp medium) and the number of Ade+ prototrophs (SCPAde medium). Median mitotic recombination frequencies (A&+ cells/Trp' cells) were determined and rates

Homologous Recombination Pathways in S. cermisiae

TABLE 1

Description and sources of disruption plasmids

Name Description Source

pL962 rad1::LEUZ disruption plasmid R. KEIL

pNKY83 rad50::hisGURA3-hisC disruption plasmid ALANI et al. (1989) pAM28 rad51::LEU2 disruption plasmid M. AKER

pSM31 rad54::LEU2 disruption plasmid D. SCHILD pSTLl1 rad55::LEUZ disruption plasmid LOVETT and MORTIMER (1987) pSM51 rad57::LEUZ disruption plasmid D. SCHILD PEI139 xrsP::URA? disruption plasmid IVANOV et al. (1994)

47

(events/cell/generation) were calculated according to the following formula: rate = (0.4343 X median frequency)/ (log N - log No), where N is the number of Trp+ cells present in the colony and No (number of initial cells) = 1 (DRAKE 1970). Neither the mating type nor the SPOI? allele affected recombination rates, therefore data from isolates differing in either of these genotypes were pooled.

Characterization of recombinants: A single Ade+ recombinant was picked from each original colony to ensure analysis of independent events; DNA was isolated (HOFFMAN and WIN- STON 1987), digested with NdeI or PstI and analyzed by South- ern hybridization to determine the nature of the recombination event. Blots of DNA digested with NdeI were probed with a radioactively labeled fragment of the TRPl gene, and blots of DNA digested with PstI were probed with a fragment of the ADE2 gene. The expected fragments from this analysis in strains transformed with pAL90-1 are shown in Figure 2B. Statistical analysis was done by using a contingency chi- squared analysis on data assembled into tables. A P value <0.05 was considered to be statistically significant.

RESULTS

Experimental system: To examine the conservative nature of recombination events a substrate based on intrachromosomal inverted repeats was constructed (RATITRAY and SYMINGTON 1994). The advantage of this substrate over direct-repeat substrates is that crossovers are constrained to occur by a fully reciprocal mechanism. Full exchanges (reciprocal crossovers) result in the inversion of the intervening DNA. These may be accompanied by conversion of the restriction enzyme mutation in the full-length repeat. Gene conversions without crossing over leave the intervening DNA in its original configuration, and are detectable by the conversion of a restriction-enzyme fill-in mutation. Half-exchanges lead to a lethal fragmentation of the chromosome, and are therefore not recoverable (see Figure 1).

The substrate utilizes two mutated alleles of the ade2 gene flanking a copy of the TRPl gene (Figure 2A). In Rad' strains recombination to adenine prototrophy occurred at a rate of 1 X events/cell/generation (Table 3). The nature of a recombination event was determined by Southern analysis of the Adet products (Figure 2B). Of 48 independent Ade+ recombinants, 50% occurred by gene conversion without reciprocal exchange, 35% by crossover and 15% by crossover asso-

ciated with conversion (Table 3), Gene conversions can occur by either a sister-chromatid or intrachromatid event, and crossovers are confined to intrachromatid events. In a similar study in bacteria, SEGALL and ROTH (1994) found that between 10 and 50% of recombinants were double crossovers (see Figure 1 ) . Although this class of events could have been distinguished, this class was not recovered in this study. We previously demonstrated that recovery of all three classes of events was strongly dependent upon the RAD52 gene product (>500@fold), whereas in a rad51 mutant strain conversions were reduced 18-fold, and crossovers (both with and without associated conversion) were reduced only 2.5-fold (RATTRAY and SYMINGTON 1994). The aim of the current study was to determine the rate and class distribution of recombination events in strains mutated for other genes in the RAD52 epistasis group.

RALI51 is epistatic to M 5 7 for inverted-repeat recombination and the repair of ionizing radiation induced DNA damage: The Rad51 amino acid sequence shows significant homology to prokaryotic RecA proteins ( ~ O U S S E K H R A et al. 1992; BASILE et al. 1992; SHI- N O W et al. 1992), yet rad51 mutants display a much less severe recombination phenotype than that of recA mutants ( ~ O U S S E K H R A et al. 1992; SHINOHARA et al. 1992; RAnRAYand SYMINGTON 1994). One possible reason for this difference is that there is functional redundancy between the Rad51, Rad55, Rad57 and Dmcl proteins, all of which show homology to RecA (KANS

and MORTIMER 1991; BISHOP et al. 1992; LOVETT 1994). Although the Dmcl protein shows the most homology to Rad51, it is only expressed during meiosis, and dmcl mutants exhibit no mitotic phenotype (BISHOP et al. 1992). To determine whether the mitotically expressed RecA-like proteins have redundant functions, the effect of mutations in RAD55 and RAD57 on inverted-repeat recombination was examined.

Initially, the rate of recombination was determined in a rad57 mutant and in a rad51 rad57 double mutant strain and compared with wild-type and rad51 strains (Table 3). Whereas recombination was only reduced 5- fold in a rad51 mutant, it was reduced 30-fold in a rad57 mutant. The rad51 rad57double mutant strain showed a fivefold reduction in recombination, indicating that


TABLE 2

W303-1A W303-1 B" W83815D W828-6A LSY4 10 U671 U672 W82&6C 1.SY369 yAR69 yAR7 1 yAR82

yAR9 1 yAR97-3.4, 10B

yAR97-9C

yAR97-4D

yARI01-5A, 6C, 8C

yAR101-9B

yAR107 yAR 1 08 yAR122-2D, 'LID, 29D

yARH6

yARl39 yAR15&10D, 43D

yAR15Cil8C

y A R 159-47A

yAR1.59-75B

yARlfiO-IC:, 19D

yAR161-2D

yAR161-32A, 42B

yARl67

yAR18453C

yAR184-2

yAR188 yAR190

MATa t?;ol-l luu2-3,112 hi.s3-11,15 urn3-1 ndo2-1 cunl-1OO MA7h trpl-l leu2-3,112 his3-11,15 urni-l ad&-1 runl-IO0 rad1::LEUZ del-ivative of W303-1A md50::hisG derivative of W'303-1 R md51::URA3 derivative of W303-1A md54::IXUZ derivative of W303-1A rad55::L.kXJ2 derivative of M'9031A rnd57::IEUZ derivative of. W303-1 B xrr2::URA3 derivative of WJO3-IB nclr2:rh~ss(~URAi-hisG; .sp113::hisG derivative of W303-1A ndc2-SA-TKP1-nd2-n dcrivative of yAR69 (transformation with pAL90) rudl::lXU2 derivative of $371 (transplacement with ~1,962) md57::UU2 derivative of yAR71 (transplacement with pSM51) (~~r2-5'A-7'RPl-(rclrZ-n derivative of yAR77 (transformation with pAL90) MAT@ ccde2::h1.sC,URAi-his(; rclrl50::AisC udr2-5'A-TRPI-(rdlr2-n (from

MATa nd~2::hbGC~KA3-hisC: mcl50::hisG nde2-SA-TRI ' l -~d~2-~1 cross o f yAR71 with W828-6A)

cpol3::hisG (from cross of yAR71 with W828-61%)

cross of yAR71 with W828-6A)

cross of yAR91 with U672)

cross of yAK9I with U672)

adQZ::hisCURAi-hi.cs(; md50::hisC ad~2-5'A-?'RPI-uclr2-n (from

MATa ndr2::hisCURAi-hisC; md55::IJXJ2 acle2-5'A-TRPI-ndr2-n (from

MATO adr2::hisGUM3-hisG r.ad55::lHJ2 ade2-5'A"l'RPl-nd~2-n (from

rnd51::L,liU2 derivative of yAR71 (transplacement with pAM28) rnd51::1J%2 derivative of yAR91 (transplacement with pAM28) MATa nd/~2::his~~C~KA3"hisC; racl51::UU2 rad57::IEU2 ade2-5'A-lWI-

rud54::L,liC12 derivative of. yAR71 (transplacement with pSM31) MATa nd~!2::hi.s~;URA3-ha.~G xr~2::URA3 ad~2-5'A-TWl-ade2-n spol3::hzsG

MA78 ndu2::hiGURA3-l~i.sG xrs2::URA3 ndr2-5'A-TRPI-ndr2-n spol3::hisG

MA79 ade2::hisGUKA3-hisC rad55::IHl2 rnd57::l.E112 rrdr2-5'A-TlU'I-

MATa ad~2::hisGUKA3-llis(;; rad55::IEUZ rnd57::LEUZ nde2-5'A-TWI-

MATa ndr2::hisG-URA3-hi.sG rndl::LLC12 rud51::LEU2 ndr2-5'A-TRI'l-

adr2-n rpol3::hisC (from cross of yAR107 with W82&6C)

(from cross o f vAK71 with LSY369)

(from cross of yAR71 with LSY369)

rtde2-n (from cross of yARI0I and W8'LMC)

nde2-n (from cross of yARlOl and W82&6C)

ndlr2-n (from cross of yARl08 with W838-15D)

d r 2 - n (from cross ofyAR108 with U671)

ndr2-n (from cross ofyARlO8 with U671)

(transplacement with pNKY83)

SA-YIWI-adr2-n (from cross of yAR1.59 with LSY410)

nclr2-n (made by transplacement of 5FOA' derivative of yAR159-47A with rad51::1JRA3 PCR fragment)

MATa nde2::hisC,UKA3-l~is(~ md51::1XU2 md54::LE(12 nde2-5'A-TKP1-

MATa ud~2::his~-URA3--his~~ t71d51::lJ~UZ T(ld54::Ik.'~l2 acl~2-5'A-TKPl-

(uie2::hisC; r(1d5O::hz~CT('RAi-3-his(; derivative of yARl60-1C:

MATa ndr2::hzsGZiKA3"hisC:~ md5l::/IRA3 rad55::IXU2 rad57::IXUZ ndr2-

MATa udp2:thisC: rad51::C'Rti3 rad5i::LE(l2 md57::1lU2 ndr2-5'A-TKPl-

T/Jd51::lEU2 derivative of yAR97-Yc (transplacement with pAM28) r~dl::Ih'CC? derivative of vAR97-9C (transplacement with pL962)

Description of yeast strains

Name Relevant genotype or description Source

TIIOMAS and ROTI-ISI'EIN (1989) TIrouAs aud ROTHSTEIN (1989) MCDONAI.L) and ROTHSTEIN (1994) MCDONAI.D and ROTIISI'EIN (1994) RATI'RAY and SYMIN(:TON (1994) MCDONAL.I) and ROI'HSTEIN (1994) M c D 0 ~ ~ r . o aud ROTHsIEIN (1994) M(:D~N.UD and ROTITSTEIN (1994) P. FIORENIIN RATIIUY and SYMINGTON (1994) RATTRAY and SYMIN(:TON (1994) This study This study RATTRAY and SYMIN(;.~ON (1994) This study

This study

This study

This study

This study

R A T T F A Y and SYMINGTON (1994) RZTI.MY and SYMINGTON (1994) This study

This study This study

This study

This study

This study

This study

This study

This study

This study

This study

This study

This study This study

" All strains used in this study are derivatives of W303-1A or W303-1B. Only markers that differ in genotype are noted.

the RAD51 gene is epistatic to the RALl57 gene. When Ade+ prototrophs from these strains were examined by Southern blot analysis to determine the distribution of recombination events (Table 3) , it was found that rad57 single mutant strains showed a 60-fold reduction in gene conversion events unassociated with a crossover when compared with the wild-type strain and a 20-fold reduction in crossovers either with or without a gene

conversion event. Both rad57 and rad51 rad57 strains showed a distribution indistinguishable from that seen in the rad51 single mutant ( P < 0.01). These results suggest that RAD51 and RAD57 function epistatically on the same recombination pathway and that RAD51 functions before RAD57.

The epistatic relationship between the RAD51 and W 5 7 genes for repair of ionizing radiation-induced

Homologous Recombination Pathways in S cereuisiar 49

Conversion Full exchange Double Recombinant Half exchange

I I No Inversion Inversion No inversion Lethal

FIGURE 1 .-Recombination between intrachromosomal inverted repeats. Thick lines denote homologous sequences. ade2-SA and ade2-n refer to the alleles used in this study. a and e refer to flanking sequences; b-d refer to intervening sequences. Only full exchanges where both pairs of flanking sequences are rejoined are viable and lead to inversion of the intervening DNA (after SEGALL and ROTH 1994).

DNA damage was also determined. It was found that rad51 mutants were more sensitive to ionizing radiation than rad57mutants (Figure 3 ) and that the rad51 rad57 double mutant was as sensitive as a rad51 single mutant. Although results from both the recombination assay and the DNA repair assay indicate that RAD51 is epistatic to RAD57, the results are qualitatively different. In the recombination assay, the phenotype of a rad51 mutant is less severe than that of the rad57 mutant, whereas the opposite is true for repair of ionizing radiation-induced damage. We suggest that this is due to the ability of alternative recombination pathways to repair a subset of spontaneous lesions that lead to recombination in the inverted-repeat assay, whereas most ionizing radiation-induced damage must be repaired via a RAD5I-dependent pathway. As ionizing radiation gen- erates double-strand breaks, it is likely that the RADSI- dependent pathway acts on these lesions, whereas the alternate recombination pathway(s) may be active on other types of DNA lesions that are generated during growth.

rad55mutants are phenotypically similar to rad57mu- tants for spontaneous mitotic recombination: Disrup- tion of the RAD55 gene resulted in a 20-fold decrease in the rate of Ade+ prototroph formation (Table 3 ) . This is of similar magnitude to the decrease seen in rad57 mutants. The rate of recombination in a rad55 rad57 double mutant was decreased 25-fold (Table 3) with no further decrease over either single mutant. Be- cause rad55 and rad57 mutants are cold sensitive (cs) for the repair of ionizing radiation (GAME and MORTI-

MER 1974; GAME 1983, 1993; LOVETT and MORTIMER 1987), we determined the effect of incubation at different temperatures on inverted-repeat recombination in the single- and double-mutant strains (Figure 4). We found that recombination in all three strains was cs, being reduced between 50- and 80-fold at 20" and only reduced 3- to 10-fold at 37". Twenty-four Adef recombinants isolated from each of these strains at 30" were examined by Southern analysis (Table 3). Comparison of the distribution of events between the rad55 and rad57 single mutants and the rad55 rad57 double mutant showed no significant differences ( P > 0.1). We also examined the distribution of recombination events in the rad55 rad57double mutant at 20 and 37" (Table 3 ) and found that all three classes of events were reduced at low temperatures. The finding that rad55 and rad57single and double mutants all have similar phenotypes suggests that these two proteins function at the same or a similar step in recombination.

A rad51 rad55 rad57 triple mutant is still proficient at spontaneous mitotic inverted-repeat recombination: We found that rad51 rad57or rad55 rad57double- mutant strains were still relatively proficient at inverted- repeat recombination (see above) as compared with a rad52 mutant, which is reduced 3000-fold (RAmRAYand SYMINGTON 1994). To determine if the presence of a single one of the mitotic RecA homologues is sufficient to promote recombination, a rad51 rad55 rad57 triple- mutant strain was constructed. Recombination in the triple mutant was reduced only five-fold, to a rate identi- cal to a rad51 rad57 double mutant strain (Table 3).

50 X . J . Rattray and 1.. S. Symington

A. INVERTED REPEAT SUBSTRATE

N P L "-

ade2-SA TRPI ade2-n

1.2 kb Ndel digest, TRP I probe PsrI digest, ADE2 probe -

"

". 2.3 kb 6.4 kb

Probe

B. CLASS I: SIMPLE GENE CONVERSION

N P

Ndel digest, TRP I probe

PsrI digest, ADE2 probe

"

2.4 kb ". ".

6.4 kb

CLASS 11: SIMPLE CROSSOVER

" ~~

NdeI digest, TRP I probe 4.6 kb ".

PsrI digest, ADE2 probe ".

5.9 kb

CLASS 111: CROSSOVER AND GENE CONVERSION

~p N P P N N P ~ 1 L

2.4 kb "

NdeI digest, TRP I probe

PsrI digest, ADE2 probe A ". 2.1 kb 5.9 kb

FIGURE 2.-Physical map of the inverted-repeat substrate. (A) Unrecombined substrate showing the expected fragments from restriction endonuclease digestion and Southern blot- ting. (B) Classes of Ade' recombinants with the fragments predicted from Southern blot analysis indicated. N, lVd~l; P, t u .

These results indicate that there is no fhctional redundancy between these genes and that RAD51 is epistatic to both RAD55 and RAD57. Furthermore, we found that a mutation in RAD51 suppressed the cs phenotype of a rad55 rnd57double mutant for inverted-repeat recombination (Figure 4). When 24 Ade' prototrophs were examined by Southern blot analysis (Table 3), it was found that the distribution was not significantly different than the distribution seen in rnd51, rad51 red57 or rad55 rad57 strains ( P > 0.1).

W 5 Z is epistatic to W 5 4 Cells with a mutation in the RAD54 gene show severe defects in the repair of ionizing radiation-induced DNA damage (GAME and MORTIMER 1974) and in spontaneous and induced mitotic recombination (SAEKI et d . 1980). This gene is a member of the RAD5, RAD16 and ShF2 gene family, all ofwhich have weak homology to DNA helicases (SCHLD et nl. 1992). Disruption of the RAD54 gene resulted in a 25-fold decrease in the rate of inverted-repeat recombination (Table 3). Southern analysis of 24 indepen-

dent Ade' recombinants (Table 3) showed a distribution of events that was significantly different from wild tvpe (1' < 0.05) and similar to that seen in rad51 mutants ( P > O.l), where the greatest reduction is in the gene conversion not associated with a crossover class. To determine the epistatic relationship of the IL41)51 and IWIl54 genes, a rad51 rlrd54 double-mutant strain was constructed. The double mutant was only reduced five-fold for inverted-repeat recombination (Table 3), indicating that RAD51 is epistatic to RAD54. The distribution of events in the double mutant was similar to that of either single mutant ( P > 0.1). Because the rad54 single mutant strain showed a similar reduction in recombination to that seen in both md55 and rad57 single mutants, and all three of these mutants showed similar epistasis with a rad51 mutant, we conclude that the IUD54 gene functions downstream of M 5 1 , perhaps at the same or similar step in recombination as IUD55 and RAD57.

Inverted-repeat recombination is reduced in rad50 and xrs2 mutants: The KA1150 and XRS2 genes are required for the repair of ionizing radiation-induced DNA damage and for meiotic recombination (GAME and MORTIMER 1974; MALONE and ESPOSITO 1981; ZAKHAROV Pt nl. 1983; AIANI et 01. 1989; MAI.ONF. et nl. 1990; IVANOV et nl. 1992). However, mutation of either gene results in increased rates of spontaneous heteroallelic recombination and proficiency at mating type switching (AIANI et nl. 1989; MALONE et al. 1990; IVANOV Pt nl. 1994). The effect of mutations in these genes on recombination between inverted repeats was determined. We found that both mutants showed a slight reduction in the production of Ade' prototrophs of threefold for the rad50 mutant and fivefold for the xn2 mutant (Table 4). Examination of the recombination events indicated a two- to threefold decrease for all classes of events in rad50 mutants and a four- to sev- enfold decrease for all classes of events in xrs2 mutants. This distribution did not differ significantly from the wild-type strain (IJ > 0.1).

We also examined a rad50 rad51 double mutant strain and found that the rate of Ade' prototroph formation was similar to a rod51 single mutant (Table 4). The distribution of recombination event5 was not significantly different from a rad51 mutant ( P > 0.1) but did differ from both wild-type and rnd50strains ( P < 0.05). These data suggest that RAD50 and RAD51 function in the same pathway.

The W Z gene identifies a second recombination pathway for inverted-repeat recombination: The KAI)1 gene is involved in excision repair of UV-induced DNA damage (see GAME 1983). The Rad1 protein forms a complex with the Rad10 protein that specifies a single- strand (ss) DNA endonuclease (TOMKINSON et nl. 1993). Although the RAD1 gene is not required for spontaneous mitotic heteroallelic recombination, rad1 mutants have been shown to have reduced levels of deletion

Homologous Recornhination I'athways in S. c w ~ ~ i s i r w

TABLE 3

Effect of md51, md55, md57 and rad54 mutations on inverted-repeat recombination

300 (24/48) 26 (3/24) 8 (6/24)

23 (3/24) 20 (9/23)E 4 ( 5 / 2 3 ) 7 (4/24)

76 (8/24) 55 (5/24)

5 (4/24) 14 (2/24)

330 ( 17/48) 140 ( I .5/24) 16 ( l2/24)

1 I O ( l4/24) 18 ( 8 / 2 3 ) I O ( 13/23) 1 1 (6/24) 6 5 (7/24) 63 (8/24) 23 ( 13/24) 95 ( 13/24)

150 (5/48) 3.5 (6/24) 8 (6/24)

.37 (7/24) 13 (6/23) 4 (.5/23)

24 ( 14/24) 87 (9/24) 52 (9/24) 12 (5/24) 68 (9/24)

" Rates arc evcnts/cell/gcner;tion; cxperimcnts were performed a t 30" unlcss othcnvisc st;~tctl; nrml)crs i n

'' Gene conversions associated with a reciprocal crossover. ' Data from three independent experiments; median value o f 81 colonies examined. ' I Data taken from RATI'KAY and SIIiIw;rox (1994). 'Distrihrttion o f events is signilicantly different from wild-type strain (P< 0.0.3). Distribution of events is not significantly different from md51 strain ( P > 0.1 ).

I' One of 24 events examined showed a complex restriction pattern.

parentheses indicate number o f et'ents in class/total events examined.

events between sequences oriented as direct repeats (KLEIN 1988; SCHIESTL. and PRAKASH 1988). We found that disruption of theRADl gene had no effect on the rate of Ade' prototroph formation (Table 4). When 24 Ade' recombinants were examined by Southern blot analysis, the distribution of events was found not to differ significantly from that seen in a wild-type strain ( P > 0.05). We next examined the epistatic relationship of the RAD1 and RA1151 genes by determining the rate of recombination in a rad1 rad51 double mutant. The rate of Ade' prototroph formation was reduced 17-fold (Table 4). Southern blot analysis of Ade' recombinants indicated a synergistic decrease in the rate of crossovers unassociated with a conversion (Table 4, class 11), which

is significantly different from a rlrd51 mutant strain (P< 0.025). These data indicate that some lesions normally processed via a IG11151dependent path\vay arc r e p a i d via a RAI11-dependent pathway.

To further investigate the relationships betwecn R A D I , RA1)50 and IUI151, double-and triple-mutant strains were constructed. The rate of Ade' prototroph formation in a rad1 rd50douhlc mutant was intermecli- ate between either single mutant (Table 4). Southern analysis of recombinants also indicated an intcrmrtliatc phenotype. However, the rate of recombination w a s rc- duced 50-fold in the triple mutant, indicating a multiplicative decrease over the r d l md51 double mutant. The distribution of recombination events i n thc triple mutant was significantlv different from that seen in rudl, rad50 and rad1 rctd50 strains (I' < 0.0.5) but was not different from a rd51 mutant (!'> 0.1).


TABLE 4

Effect of rad50, xrs2 and radl mutations on inverted-repeat recombination

Rate of Ade+ products (X lo7)

Total Ade+ Class I Class I1 Genotype

Class I11 ( X 106) conversions crossovers both

RAD 100 500 (24/48) 350 (17/48) 150 (7/48) rad5G" 37 162 (21/48) 162 (21/48) 46 (6/48) xrsT 19 '72 (9/24) 87 (11/24) 30 (4/24) rad51 22 26 (3/24) 140 (15/24) 55 (6/24) rad50 rad51"' 20 34 (4/24) 92 ( 1 1/24) 74 (9/24) rad 1 ' I 110 412 (9/24) 275 (6/24) 412 (9/24) radl rad51".' 6 12 (5/24) 10 (4/24) 38 (15/24) radl rad50' 49 264 (13/24) 123 (6/24) 103 (5/24) radl rad50 rad51' 2 8 (9/22)" 6 (7/22) 5 (6/22)

Data presented as in Table 3. I' Distribution of events is not significantly different from wild-type ( P > 0.1). "Distribution of events is not significantly different from rad51 ( P > 0.1). ' Distribution of events is significantly different from wild-type ( P < 0.05). "Two of 24 events examined had a complex restriction pattern.

DISCUSSION

A chromosomal inverted-repeat substrate has been used to investigate the genetic control of homologous recombination in S. cereuisiae. The data presented here provide evidence for the existence of multiple pathways for spontaneous mitotic recombination. This conclu- sion is based on the following observations. (1) Whereas rad52mutants are reduced 3000-fold for inverted repeat recombination, rad51 mutants are only reduced 5-fold (RATTRAY and SYMINGTON 1994). (2) Single mutations in m 5 4 , RAD55 or RADS7 result in a 20- to 30-fold decrease in recombination but reduce recombination only !?-fold in combination with a rad51 mutation. These data indicate that all four of these genes function in the same recombination pathway, that RAD51 functions before the other three and that an alternative pathway must function in the absence of RAD51. (3) rad55 and rad57 mutants, as well as double mutants, are cs for inverted-repeat recombination. (4) Disruption of all three mitotic RecA homologues results in a recombination phenotype indistinguishable from a rad51 single mutant, indicating a lack of functional redundancy. The triple mutant is not cs for recombination, further supporting an early role for Rad51 in recombination. (5) Although disruption of the R A D 1 gene does not affect inverted-repeat recombination, rad1 rad51 double mutants show a synergistic decrease indicating that some of the M5l-independent recombination is processed via a RADldependent pathway. (6) Whereas rad50 and xrs2 mutants show elevated rates of heteroallelic recombination, these mutants have reduced levels of inverted-repeat recombination.

The genes in the RAD52 epistasis group were initially identified by the sensitivity of mutants to ionizing radiation (GAME and MORTIMER 1974). rad52 mutants are the most sensitive to ionizing radiation and are epistatic

to all other genes in this group (MCKEE and LAWRENCE

1980). Although all of the mutants are sensitive to ionizing radiation, considerable heterogeneity exists between members of this group when other phenotypes related to recombinational repair are examined. For example, one expectation for a mutant defective in recombinational repair is that haploids and diploids should be equally sensitive to ionizing radiation. rad51, rad52 and rad54 mutants share this property, but xrs2, rad50, rad55 and rad57 mutants all show greater resis- tance to ionizing radiation in diploids than in haploids (SAEKI et al. 1980; LOVETT and MORTIMER 1987; ABOUS

SEKHRA et al. 1992; IVANOV et al. 1992). Although this is referred to as diploid-specific repair, in the case of rad55 mutants it has been shown to be because of mating type heterozygosity rather than ploidy (LOVETT and MORTI- MER 1987).

The assay commonly used to measure mitotic recombination is the rate of prototroph formation between heteroalleles of an auxotrophic marker in diploids. These events occur primarily by gene conversion unassociated with crossing over (ROMAN 1957; KAKAR 1963; HURST and FOGEL 1964; GOLIN and ESPOSITO 1981). rad51 and rad52 mutants show the greatest defects in spontaneous and induced heteroallelic recombination, whereas rad54, rad55 and rad57mutants show a modest reduction with a substantial induction by y-rays. rad50 and xrs2 mutants share the property of elevated rates of spontaneous heteroallelic recombination. Given that rad50, xrs2, rad55 and rad57 mutants show diploid specific repair, it is possible that recombination in these mutants would be reduced further in haploids. Recom- bination can be measured in haploid strains that contain gene duplications. These are generally arranged as intrachromosomal direct repeats, intrachromosomal inverted repeats or on heterologous chromosomes. Mu-

Homologous Recombination Pathways in S. cerevisiae 53

tation of RAD52 yields a recombination-defective phenotype in all of these assays (JACKSON and FINK 1981; KLEIN 1988; SCHIESTL and PRAKASH 1988; AGUILERA and KLEIN 1989; BAILIS and ROTHSTEIN 1991; DORNFELD and LMNGSTON 1992), but the effects of other genes in the RAD52 epistasis group have previously only been analyzed for direct-repeat recombination. Deletion events between direct repeats (pop-outs) are increased in rad51, rad54, ,rad55 and rad57 mutants and occur at wild-type frequency in rad50 mutants (MCDONALD and ROTHSTEIN 1994; H. KLEIN, personal communication). Thus, the genes in the RAD52 group can be further subdivided based on their effects on direct repeat recombination.

Our results using a chromosomal inverted-repeat recombination assay show a similar heterogeneity between the rad mutants. rad52 mutants show >3000-fold decrease in the rate of Ade' prototrophs, and all classes of recombinant products are reduced equally (RATTRAY and SYMINGTON 1994). Mutation of RAD54, RAD55 or RAD57 results in a 20- to 30-fold decrease in the rate of recombination, whereas rad51 mutants show only a 5-fold reduction (Tables 3 and 4). In all of these singly mutated strains, there is a significant decrease in gene conversion events unassociated with a crossover, compared with wild-type strains, and the distribution of events is not significantly different between all four mutant strains. rad50 and xrs2 mutants show a three- to five-fold reduction in recombination, and the distribution of events is the same as that observed in the wild- type strain. Therefore, using the inverted-repeat assay, the mutants can be divided into three groups. rad52 mutants occupy a unique position in showing the greatest reduction in recombination; rad51, rad54, rad55 and rad57 mutants share the property of reduced recombination, specifically of gene conversion events unassociated with crossing over, and rad50 and xrs2 mutants show a modest reduction in all classes of recombination events. Although rad51 mutants show a less severe recombination defect than rad54, rad55 and rad57, they are grouped together based on the phenotype of double and triple mutants. We found that rad51 rad54, rad51 rad57 double mutants and rad51 rad55 rad57 triple mutants have a phenotype indistinguishable from a rad51 single-mutant strain. The simplest interpretation of these data is that the RADS1 gene product functions before the RAD54, RAD55 or RALl57 gene products, that one (or more) alternative pathway(s) can be used in the absence of RAD51 and that once recombination intermediates have been channelled into the RADS1 pathway they can no longer be acted upon by an alternative pathway. In this scenario the alternative pathway(s) must normally process only -5% of the lesions in a wild-type cell, based upon the 20- to 30-fold reduction in recombination by mutants in the downstream genes, but can process up to 20% of the lesions in a rad51 mutant. Although mutations in all four of

these genes reduce gene conversions not associated with a crossover more drastically than crossovers (either with or without an associated conversion), crossovers are also reduced. Further support for an earlier role in recombination for RAD51 comes from analysis of the cs recombination phenotype of rad55 and rad57 mutants. Mutation in either of these genes have previously been shown to result in a cs repair phenotype (GAME and MORTIMER 1974; GAME 1983,1993; LOVE= and MORTI- MER 1987), and we show here that they are also cs for inverted-repeat recombination. We also find that there is no further reduction in the double mutant, indicating that it is likely that these two proteins function at the same step in recombination. However, we find that the cs phenotype is largely suppressed in the presence of a rad51 mutation (Figure 4).

The modest reduction in recombination observed in rad51 mutants raised two questions. First, is the intrachromosomal inverted-repeat assay representative of recombination between homologues, and second, is there functional redundancy between RecA homologues? Al- though recombination between heteroalleles occurs primarily by gene conversion unassociated with crossing over, 50% of the recombination events between inverted repeats occur by crossing over. Although mutation of RAD51 results in only a 5-fold reduction in recombination, gene conversion unassociated with crossing over is reduced 18-fold. Spontaneous heteroallelic recombination has been shown to be reduced -20- fold in rad51 mutants (SAEKI et al. 1980; ABOUSSEKHRA

et al. 1992; SHINOHARA et al. 1992). If only the class of events corresponding to gene conversions is considered, the effects of a RAD51 mutation are quite similar in these two assays.

Four yeast genes have been shown to encode homologues of the E. coli RecA protein, RAD51 (ABOUS

SEKHRA et al. 1992; BASILE et al. 1992; SHINOHARA et al. 1992), DMCl (BISHOP et al. 1992), RAD55 (LOVETT 1994) and RAD57 (KANS and MORTIMER 1991). Of these, Rad51 shows the greatest homology with RecA and has some of the expected biochemical properties, including DNA-dependent ATPase and nucleoprotein filament formation on duplex DNA (OGAWA et al. 1993a,b). We found that a rad51 rad55 rad57 triple- mutant strain had the same rate of recombination as the rad51 single mutant. Thus, the mitotic RecA homologues are not functionally redundant in this assay and appear to function in the same recombination pathway (see above). The lack of redundancy in this assay was not unexpected because mutation of any of the mitotic RecA homologs yields a y-radiation-sensitive phenotype, and mutation of any of the four genes results in sporulation and spore viability defects (PETES et al. 1991). This suggests that perhaps the many roles of RecA in bacteria are partitioned in yeast among several different proteins or that the proteins function together in a complex. The formation of a complex would be


consistent with the cs repair defect of rad55 and rad57 mutants. Furthermore, because there are still considerable levels of mitotic recombination in the absence of all three gene products, it suggests the presence of (an)- other RecA-like function (s). It is possible that the DMCl gene is expressed in a rad51 mutant, and either one of these genes is sufficient for mitotic recombination.

The role of the RADl gene was investigated because mutation of this gene reduces intrachromosomal pop- out events by up to 10-fold, although it is not required for heteroallelic recombination (KLEIN 1988; SCHIESTL and PRAKASH 1988; THOMAS and ROTHSTEIN 1989). We found that radl mutants do not affect the rate of intrachromosomal inverted-repeat recombination, and the distribution of events is the same as the wild-type strain. This contrasts with the results of AGUILERA and KLEIN (1989) who found a decrease in gene conversion tract length and decreased association with crossing over in radl mutants. Although gene conversion tract length cannot be estimated using the ade2 inverted repeat, altered rates of crossing over are readily detected. The reason for the different results obtained is not clear but could be explained by the use of different radl alleles or chromosome context effects.

We have shown that radl rad51 mutants are more recombination deficient than a rad51 single mutant strain, indicating that Radl must be able to act on lesions that are normally processed by the EliiD51 pathway. This interpretation is consistent with the results of MONTEL~ONE d aL. (1988) who proposed that Radl processes lesions generated in hyper-recombination rad31 02 mutants into a RALl52-dependent pathway. The most pronounced defect seen in rudl rad51 strains is a synergistic decrease in crossovers, and the major product recovered is crossovers associated with a gene conversion. The RAD1 pathway cannot be functioning by bypassing the requirement for RAD51 and chmnel- ling the lesions further downstream in that pathway, as in this case rad51 rad57 double-mutant strains would have the phenotype of a rad57 single mutant. It should be noted that the RADl pathway does not represent the only alternative source of recombination events, because recombination in a radl rad51 double-mutant strain is still 250-fold higher than recombination in a rad52 mutant. We cannot determine whether the RADl pathway requires the RAD52 gene, because the level of recombination in a rad52 mutant is at the limits of detection in our assay, and we would be unlikely to distinguish any synergistic decreases.

In contrast to the hyper-recombination phenotype seen in mitotic heteroallelic recombination, we find that inverted-repeat recombination is slightly reduced in both rad50and xrs2strains. The distribution of events is similar to that seen in a wild-type strain. A rad50 rad51 double-mutant strain showed no further reduction in the overall rate of prototroph formation, although the pattern of' events recovered most resembled a rad51

mutant. Furthermore, a radl rad50 rad51 triple mutant strain showed an even greater reduction in the rate of prototroph formation than did a rudl rad51 double- mutant strain. One possible explanation for this observation is that the W 5 0 gene product acts early, on a specific subset of spontaneous lesions, and that these are then channelled into either of two pathways. One pathway is acted on by Rad51 or by Radl in a rad51 mutant. The second pathway operates inefficiently in wild-type cells but accounts for some of the recombination events observed in radl rad51 mutants. Thus rad50 mutants have a weak hypo-recombination phenotype because they act on only a subset of DNA lesions. How- ever, a multiplicative effect is observed when combined with mutations in RADl and RAD51 because part of the RAD1 RAD51-independent pathway(s) is inactivated. The rationale for placing RAD50 upstream of RAD51 is based on the different phenotype compared with rad54, rad55 and rad57mutants and also the meiotic data that indicate a role before the initiation of recombination (MALONE and ESPOSITO 1981).

There are several possible interpretations of the differing recombination phenotypes of rad50 and xrs2 on heteroallelic and inverted-repeat recombination. Hetero- allelic recombination must occur by interaction between homologous chromosomes in diploids, whereas inverted-repeat recombination occurs by intrachromatid or sister chromatid interactions (collectively referred to as intrachromosomal events). It is possible that the RAD50 and XRS2 genes are involved in regulat- ing interchromosomal us. intrachromosomal events. The observation that rad50 and xrs2 haploids lack G2 repair (sister chromatid repair) but show diploid specific repair could be interpreted as retention of the ability for interchromosomal but not intrachromosomal interactions. These could be competing processes, and in the absence of a system for intrachromosomal recombination the opportunity for interchromosomal interactions may be increased, resulting in a hyper-recombination phenotype for heteroallelic recombination and hypo-recombination phenotype in intrachromosomal assays. The balance between inter- and intrachromosomal events may also be influenced by heterozygosity at the MA7 locus in diploids (KADw and H ~ T W E L I . 1992).

We present a model based on our data in Figure 5. It is clear that the vast majority of recombination in this system is dependent upon the W 5 2 gene, because rad52 mutants recombine at 0.03% wild-type levels, and the majority of Ade+ prototrophs recovered arise by a nonrecombinational mechanism (RAnuyand S W I N G - TON 1994). We cannot be certain that RAD52 initiates the pathway and its placement is merely to indicate the importance of this gene in recombination. Meiotic experiments indicate a late role in recombination (MA- LONE and ESPOSITO 1981), and a study of the processing HO-induced double-strand breaks in mitotic cells

Homologous Recombination Pathways in S. cereuisiae 55

Repaired product (Mostly mutagenic)

FIGURE 5.-Model for multiple pathways of homologous recombination between inverted repeats in yeast. The vast majority of recombination to produce Ade+ prototrophs is dependent upon the RAD52 gene. Many of the rare events recovered in rad52 mutants showed no physical alteration of the unrecombined substrate. We cannot determine when the RAD52 gene product functions, so its placement in the beginning of the pathway is only meant to denote the RAD52 dependence of recombination events. RAD50 and XRS2 appear to function early in recombination before commitment to a recombination pathway. Because rad50 and xrs2 mutants are only marginally deficient for recombination, it is likely that many lesions bypass the requirement for these gene products. Most of the recombination events appear to be promoted by a RAD51 dependent pathway, which channels lesions downstream to be acted upon by the RAD54, RAD55 and RAD57 gene products. Mutation of any of the four genes in this pathway reduces gene conversions to a greater extent than crossovers, thus we assume that this pathway resolves recombination events primarily (but not exclusively) as gene conversions. The R A D 1 gene is able to repair some of the lesions normally processed by the RAD51 pathway, although it may not function in wild-type cells. There is still 250-fold more recombination in radl rad51 cells than in rad52 cells, indicating that there must be at least one other pathway, denoted here as X. The principal product seen in these cells are gene conversions associated with a crossover.

showed that Rad50 acts before Rad52 (SUGARAWA and HABER 1992). RAD50 and XRS2 function early in meiotic recombination, and our data support the idea that RAD50 (and by analogy, XRS2) may function before the commitment to a recombination pathway. These genes are clearly not essential for most inverted-repeat recombination and may only be required for a subset of DNA lesions. The RAD51 gene appears to function early in recombination and channels lesions into a RAD54, RAD55 and RAD57dependent pathway, which results primarily (although not exclusively) in gene conversions. In the absence of the RAD51 gene, alternative pathways can act upon these lesions and result primarily in crossovers. The RAD51 pathway appears to be able to repair some of these lesions in the absence of RAD51 but is probably not used in its presence. Because radl rad51 double mutants, as well as mutants downstream of RADSZ, still recombine at rates much greater than do rad52 mutants, there is clearly another, as yet unidentified, pathway for recombination.

We thank M. AKER, E. IVANOV, R. KEIL, N. KLECKNER, R. ROTHSTEIN and D. SCHILD for gifts of plasmids and strains. This work was supported by grants from the National Institutes of Health (NIH) (GM- 41784) and the American Cancer Society (NP-73424) and in part by a grant from the IRMA T. HIRSCHI. Trust. L.S.S. is a Leukemia Society of America Scholar. A.J.R. was supported in part by NIH training grant (NIAID-AI-07161).

LITERATURE CITED

ABOUSSEKHRA, A,, R. CHANET, A. ADJIRI and F. FABRE, 1992 Semi- dominant suppressors of Srs2 helicase mutations of Saccharomyces cereuisiae map in the RAD51 gene, whose sequence predicts a

protein with similarities to procaryotic RecA proteins. Mol. Cell. Biol. 1 2 3224-3234.

AGUILERA, A,, and H. L. KLEIN, 1989 Yeast intrachromosomal recombination: long gene conversion tracts are preferentially ass@ ciated with reciprocal exchange and require the R A D 1 and RAD3 gene products. Genetics 123: 683-694.

AHN, B.-Y, and D. M. LMNGSTON, 1986 Mitotic gene converstion lengths, conconversion patterns and the incidence of reciprocal recombination in a Saccharomyces cereuisiae plasmid system. Mol. Cell. Biol. 6: 3685-3693.

A I A N I , E., S. SUBBIAH and N. KLECKNER, 1989 The yeast RAD50 gene encodes a predicted 153-kd protein containing a purine nucleotide binding domain and two large heptad-repeat regions. Genetics 122: 47-57.

BAILIS, A. M., and R. ROTHSTEIN, 1991 A defect in mismatch repair in Saccharomyces cereuisiae stimulates ectopic recombination between homeologous genes by an excision repair dependent process. Genetics 126: 535-547.

BASILE, G., M. AKER and R. K. MORTIMER, 1992 Nucleotide sequence and transcriptional regulation of the yeast recombinational repair gene RAD51. Mol. Cell. Biol. 12: 3235-3246.

BISHOP, D. R, D. PARK, L. Xu and N. KLECKNER, 1992 DMCI, A meiosis-specific yeast homolog of E. coli recA required for recombination, synaptonemal complex formation, and cell cycle pro- gression. Cell 69: 439-456.

DORNFELD, K. and D LMNCSTON, 1992 Plasmid recombination in a rad52 mutant of Saccharomyces cereuisiae. Genetics 131: 261-276.

D m , J. W., 1970 The Molecular Basis of Mutation. Holden-Day, San Francisco, CA.

ENGEBRECHT, J., J. HIRSCH and G. S. ROEDER, 1990 Meiotic gene conversion and crossing over: their relationship to each other and to chromosome synapsis and segregation. Cell 62: 927-937,

GAME, J. C., 1983 Radiation sensitive mutants and repair in yeast, pp. 109-139 in Yeast Cmetirs: Fundamental and Applied Aspects, edited by J. F. T. SPENCER, D. SPENCER and A. R. W. SMITH. Springer-Verlag, New York.

GAME, J. C., 1993 DNA double-strand breaks and the RAD5@RALl57 genes in Saccharomyces. Semin. Cancer Biol. 4 73-83.

GAME, J., and R. K. MORTIMER, 1974 A genetic study of X-ray sensitive mutants in yeast. Mutat. Res. 24: 281-292.

COLIN, J. E. and M. S. ESPOSITO, 1981 Mitotic recombination: mismatch correction and replicational resolution of Holliday struc-


tures formed at the two-strand stage i l l Snrrharomyres t . r rp~ i . t i~e . Mol. Geu. Genet. 183: 252-263.

HOFFMAN, C . , and F. Wmsroh., 1987 A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for trausfor- mation of Esrhm'chia rob. Geue 57: 262-272.

HOI.I.IIM\', R., 1964 A mechanism for gene conversion i n fungi. Genet. Res. 5: 284-SO4.

HLIRSI, D. D., and S . Fo(:EI., 1964 Mitotic recombination aut1 heteroallelic repair i u Sarrhwovyu\ rrrr?jtsiar. Genetics 50: 435- 458.

110, H., Y. FL"I).A, K. Mt1wr.A a d A. KIML~KA, 1983 Transformatiou of intact yeast cells treated with alkali cations. J. Bacteriol. 153: 163-168.

Iw\ruov, E., V. KOKOI.EV and F. FAHKE, 1992 XILS2, a DNA repair geue of Sarrhnnnoyre~ rf?#i.siae, is needed for meiotic rccombina- tion. Genetics 132: 651-664.

1994 Mutations in XRSZ and KAr150 delay but do not prevent mating-type switching in Saccharomycu.y t.mmiriar. Mol. Cell. Biol. 14: 3414-3425.

J . U : K X ~ , J. A., and G. R. FINK, 1981 Gcne conversion between dupli- cated genetic elements i n yeast. Nature 292: 306311.

KAnx, L. C . and I,. H. HAR'I'UTI I . , 1992 Sister chromatids are pre- ferred over homologs as substrates for recornhinational repair in Sacchammycus cermisiae. Genetics 132: 387402.

KAKAR, S. N., I963 Allelic recombination and its relation to recomhination of outside markers. Genetics 48: 957-966.

KANS,.!. A. and R. K. MORTIMER, 1991 Nucleotide sequence of the RAD57 gene of .Sacd~,aromyrer renwiszar. Gene 105: 139-1 40.

KI.EIN, H. L., 1988 Different types of recombination eveuts are corn trolled by the R A D Z and RAD52 geues of . krhn?otny tn rprmisiar. Genetics 120: 367-377.

Lo\%TI, S. T., 1994 Sequence of the M 5 5 gene o f Sarchuromyres cmmzsiar: similarity of RAD55 to prokaryotic RecA and other RecA-like proteins. Gene 142: 103-106.

I.ov~;TT, S . T. and K. K. MORTIMIX, 1987 Characterizatiou of n u l l mutants of the KAD55 geue of Sarrharomya rwmisiau: effects of temperature, osmotic strength and mating type. Genetics 116: 547-553.

MAILINE, R. E. and R. E. ESPOSITO, 1981 Recombinationless meiosis in Saccharomym r m r ? ~ i ~ i ~ ~ . Mol. Cell. Biol. 1: 891-901.

MAI.oN~., R. E., T. WARD, S. LIN a n d J . WARIN(,, 1990 The RAD50 gene, a member of the douhle straud break repair epistasis group, is not required for spontaneous mitotic recombination in yeast. Curr. Genet. 18: 11 1-1 16.

MCDOYAI,~, J. P. and R. ROTIISTEIN, 1994 Unrepaired heteroduplex DNA in Sar.rharomycu,t ~WYUZ.SIIC is decreased in K4DZ MD52-independent recombination. Geuetics 137: 393-405.

McK~F:, R. H. a d C . U'. LAWRLNLII, 1980 ( h e t i c analysis of y-ray mutagenesis i u yeast. I l l . Double-mutant strains. Mutat. Res. 70: 37-48.

MESEI.SON, M. and C. R\L)I)IN(;, 1975 A geueral model for recombi- natiou. Proc. Natl. Acad. Sci. USA 72: 358-361.

MII.NF,, (;. and D. U'F.A\XR, I993 Dominant uegative alleles of W 5 2 reveal a DNA repair/reconlbination complex iucludiug Rad51 and Rad52. Geues. Drv. 7: 1755-1765.

MOF;TFI.ONI:., B. A,, M. F. Hot.k3TRr\\lld R. E. Mt\I,ONb', 1988 spouta- neolts mitotic recombination iu yeast: the hyper-recomhinatiollal reml Inutations are alleles of' the K403 gene. Genetics 119: 289- 301.

OCAWA, T., A. SHINOHARA, A. NARW ANI , T. I K I ~ , \ , X. YL rl nl., 199% Rec.4 like recombination proteins in eukaryotes: functions Of

&E51 and RAD52 genes of SnrrhnromyrPs t.prr?Jisiar. Cold Spring Harhor Symp. Quant. Biol. 58: 109-137.

OGAU~A, T., X. Ycl, A. SHINOIIAKA and E. H. E(;k,iMs\N, 199% Similar-

IVANOV, E. I.., N. SU(;AM'A&\, C. 1. WHITE, F. FA~RF. a1ld.1. E. HABER,

ity of the yeast Rad51 filament to the bacterial RecA filament. Science 259: 18961899.

PRIES, T. D., R. E. MALONE and L. S. SYMINGTON, 1991 Recomhina- tion in yeast, pp. 407-521 in The Mokmlar and Cellular Biology of the Yeast Saccharomycm: Genome Dynamics, Protein Synthesis and Entlgrrzcr, edited byJ. R. BROACH, J. R. PRING1.E. and E. W. JONES. Cold Spriug Harbor Laboratory Press, Cold Spring Harbor, NY.

~ \ l ) l ) l N G , C . , 1982 Homologous pairing and strand exchange in genetic recombination. Annu. Rev. Genet. 16: 405-457.

RVL'TMY, A. J., and L. S. SWINGTON, 1994 Use of a chromosomal inverted repeat to demonsfrate that the RAD51 and RAD52genes of Sacchnromycp.~ cprmisiae have different functious in mitotic re- combiuation. Genetics 138: 587-595.

K ~ ~ : K M I I . I . , B. and G. S. ROEDER, 1990 Meiosis in asvnaptic yeast. Genetics 126: 563-574.

ROMAN, H. L., 1957 Studies of recombination in yeast. Cold Spring Harbor Symp. Quaut. Biol. 21: 175-183.

ROMAN, H. and M. RUZINSKI, 1990 Mechanisms of gene conversion in Saccharomyces cermisiae. Genetics 124: 7-25.

ROTHSTEIN, R. J., 1983 One-step gene disruption in yeast. Methods Enzymol. 101: 202-21 1.

SAF.KI, T.. I . M~C:HIUA and S. NAKAI, 1980 Genetic control of diploid recovely after y-irradiation in the yeast Sarcharomyces rmmisiae. Mutat. Res. 73: 251-265.

SCHIESTI., R. and S . PRAuw, 1988 RADl, an excision repair gene of Saccharomyces cermisiae, is also involved in recombination. Mol. Cell. Biol. 8: 3619-36'26.

Scm.L), D., 8. J. GIASSNER, R. K. MORTIMER, M. CARLSON and B. C. LU'R~NT, 1992 Identification of RAD16, a yeast excision repair gene homologous to the recornhinational repair gene RAD54 and to the SNF2gene involved in transcriptional activation. Yeast 8: 385-395.

SEC:AI.I., A. M., and J. R. RorH, 1994 Approaches to half-tetrad analysis iu bacteria: recombination between repeated, inverse-order chromosomal sequences. Genetics 136: 27-39.

SHERMAN, F., G. FINK and J. HICKS, 1986 Methods in Yeast Genetin. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

SHIKOHARA, A,, H. OGAWA and T. OGAWA, 1992 Rad51 protein involved in repair and recombination in S. cermisiar is a RecA-like protein. Cell 69: 457-470.

SMITII, G. R.. 1989 Homologous recombination in E. coli: multiple pathways for multiple reasons. Cell 5 8 807-809.

SUGAW.,\M, N., and J. E. HAWR, 1992 Characterization of douhle- strand break-induced recombination: homology requirements and single-stranded DNA formation. Mol. Cell. Biol. 12: 56% 575.

Szosr,w, J. W., T. I,. ORR-WEAVER, R. J. ROTHSTEIN and F. W. STAHI., 1983 The double-strand-break repair model for recombination. Cell 33: 25-35.

THOMAS, B. J. and R. ROTHSTEIN, 1989 The genetic control of direct- repeat recombination in Saccharomyces: the effect of rad52 and m d Z on mitotic recomhination at GALIO, a transcriptionally reg- ulated gene. Genetics 123: 725-738.

TOMKINSON, A. E., A. J . BARDWEJ.~., L. BARDWEL.~., N. J . TMPE and E. <:. FRIEDEERC,, 1993 Yeast DNA repair and recombination proteins Rad1 and Rad10 constitute a single-stranded-DNA endo- nuclc-ase. Nature 362: 860-862.

WINS TO;^, F., F. CHUMLFX and G. R. FINK, 1983 Eviction and transplacement of mutant genes in yeast. Methods. Enzymol. 101: 21 1-228.

Z,\KHAROV, I. A., <;. V. KAsslNOVh and s. v. KOVr\I,TZ<)VA, 1983 Intra- genic mitotic recombination induced by ultraviolet and gamma rays in radiosensitive yeast mutants. Genetika 19: 49-57.

ZHIFLIS, B. R., A. D. MCWII.I.IAMS, Y.-H. LIN, M. F. HOEKSTRA and R. L. &x,, 1990 Genetic control of RNA polymerase I-stirnu- lated recombination i n yeast. Genetics 126: 41-52.

Communicating editor: S . JINLS-ROBERTSON

Documents

Multiple Pathways for Homologous Recombination in ...46 A. J. Rattray and L. S. Symington that are required for recombination in Saccharomyces cerevisiae, it has not been clearly demonstrated