Structure and Function of DNA Repair Nuclease

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Structure and function of nucleases in DNA repair: shape, grip and blade ofthe DNA scissors

Tatsuya Nishino1 and Kosuke Morikawa*,1

1Department of Structural Biology, Biomolecular Engineering Research Institute (BERI), 6-2-3 Furuedai, Suita, Osaka 565-0874,Japan

DNA nucleases catalyze the cleavage of phosphodiesterbonds. These enzymes play crucial roles in various DNArepair processes, which involve DNA replication, baseexcision repair, nucleotide excision repair, mismatchrepair, and double strand break repair. In recent years,new nucleases involved in various DNA repair processeshave been reported, including the Mus81 : Mms4 (Eme1)

complex, which functions during the meiotic phase andthe Artemis : DNA-PK complex, which processes a V(D)Jrecombination intermediate. Defects of these nucleasescause genetic instability or severe immunodeficiency.Thus, structural biology on various nuclease actions isessential for the elucidation of the molecular mechanismof complex DNA repair machinery. Three-dimensionalstructural information of nucleases is also rapidlyaccumulating, thus providing important insights into themolecular architectures, as well as the DNA recognitionand cleavage mechanisms. This review focuses on thethree-dimensional structure-function relationships ofnucleases crucial for DNA repair processes.

Oncogene (2002) 21, 9022 9032. doi:10.1038/sj.onc.1206135

Keywords: DNA repair; nuclease; metal-dependentcleavage; protein-DNA interaction; structure-functionrelationships

Introduction

Quality control of genetic material is a functionconserved in all living organisms. DNA suffers frommany environmental stresses, including attacks by

reactive oxygen species, radiation, UV light, andcarcinogens, which modify the DNA. In addition,there are intrinsic errors and unusual structures, whichare formed during replication or recombination, andthey must be corrected by the various repair proteinmachineries to avoid alterations of the base sequencesor entanglement of the DNA. These DNA repairproteins may function independently, but in manycases, they form complexes to perform more efficientrepair reactions. In the repair complexes, nucleasesplay important roles in eliminating the damaged or

mismatched nucleotides. They also recognize thereplication or recombination intermediates to facilitatethe following reaction steps through the cleavage ofDNA strands (Table 1).

Nucleases can be regarded as molecular scissors,which cleave phosphodiester bonds between the sugarsand the phosphate moieties of DNA. They contain

conserved minimal motifs, which usually consist ofacidic and basic residues forming the active site.These active site residues coordinate catalyticallyessential divalent cations, such as magnesium,calcium, manganese or zinc, as a cofactor. However,the requirements for actual cleavage, such as the typesand the numbers of metals, are very complicated, butare not common among the nucleases. It appears thatthe major role of the metals is to stabilize inter-mediates, thereby facilitating the phosphoryl transferreactions. Cleavage reactions occur either at the endor within DNA, and thus DNA nucleases arecategorized as exonucleases and endonucleases, respec-

tively (Figure 1). Exonucleases can be furtherclassified as 5 end processing or 3 end processingenzymes, according to their polarity of consecutivecleavage.

This review describes the three-dimensional (3D)structural views of the actions of various nucleasesinvolved in many DNA repair pathways. The rapidlyaccumulating genomic, biochemical and structural datahave allowed us to classify various nucleases intofolding families. In general, the nucleases involved inDNA repair recognize the damaged moiety through theremarkably large deformation of DNA duplexes, andthus in terms of their DNA recognition mode, theyapparently differ from the sequence-specific endonu-

cleases, such as the restriction enzymes. The active sitesof DNA repair nucleases have some similarity withother nucleases, including the metal-coordinatingresidues; however, they also display pronounceddiversity.

Nucleases in various categories of DNA repair

Replication

DNA polymerase replicates a new strand of DNA, thesequence of which is complementary to the templateDNA. Most DNA polymerases in prokaryotes and

eukaryotes are composed of two different enzymes, a*Correspondence: K Morikawa; E-mail: [email protected]

Oncogene (2002) 21, 9022 9032 2002 Nature Publishing Group All rights reserved 0950 9232/02 $25.00

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polymerase and an exonuclease, encoded within thesame polypeptide, but sometimes they are formed bydifferent subunits. The exonuclease degrades misincor-porated DNA strand in the 3 to 5 direction (Figure 2)(reviewed in Shevelev and Hubscher, 2002). Deletionof these proofreading nucleases results in lethal orstrong mutator phenotypes in bacteria (Fijalkowskaand Schaaper, 1996) and in yeast (Morrison et al.,1993), and causes cancer in mice (Goldsby et al.,2001).

The removal of Okazaki fragments is anotherimportant process in replication. This DNA : RNAhybrid is required to initialize DNA polymerization,

but once the replication starts, it is rapidly degraded.

Most of the Okazaki fragments are eliminated by

RNaseH, enzyme ubiquitously present in all livingorganisms. RNaseH produces nicks in the RNA regionof Okazaki fragments (Figure 2). In eukaryotes and inarchaea, FEN1 endonucleases also participate in theremoval of Okazaki fragments (reviewed in Lieber,1997). FEN1 is a multi-functional enzyme. In additionto the 5 to 3 exonuclease activity to remove theOkazaki fragments, the enzyme can also generate anincision at the junction point of a 5 flap DNAstructure. This latter activity is required to eliminatenon-homologous tails in base excision repair and inrecombination intermediates.

The replication process is stalled by various modesof DNA damage. Upon the halt of fork progression,

the DNA polymerase and other protein complexesabandon the replication fork. The remaining fork mustbe processed by various fork-specific protein machi-neries. The most notable protein among them isMus81, which was recently found as a new fork/junction specific endonuclease (Boddy et al., 2000,2001; Interthal and Heyer, 2000; Kaliraman et al.,2001; Mullen et al., 2001). Genetic and biochemicalanalyses have revealed that this endonuclease iscompletely conserved in eukaryotes, while its homologhas been found in archaea. The loss of Mus81 in yeastcauses UV or methylation damage sensitivity (Interthaland Heyer, 2000) and defects in sporulation (Mullen et

al., 2001).

Table 1 Nucleases involved in DNA repair

Prokaryote/Bacteriophage Archaea Yeast Mammals

ReplicationProofreading PolI, II PolB, D Pold, e, g Pold, e, g

DnaQ

Okazaki fragment processing RNaseH RNaseHII RNaseH RNaseHFEN1 FEN1Dna2

Replication fork cleavage Hef Mus81 Mus81(+Mms4[Eme1])a,b Wrn

Base excision Repair EndoV APN1Abasic site processing EndolIV HAP1[APE,APEX]b

ExoIIIMismatchrepair MutH

Nucleotide excision repair5 processing UvrC(+UvrB)a Rad1(+Rad10)a XPF(+ERCC1)a

3 processing UvrC Rad2 XPGShort patch repair Vsr

Double strand break repair

End processing RecB(+RecCD)a

Dna2SbcD(+SbcC)a Mre11(+Rad50)a Mre11(+Rad50)a Mre11(+Rad50)a

RecJExoVII[RecE]b

ExoI[SbcB]b Artemis(+DNA-PK)a

Holliday junction resolvase RuvC Ccell[Ydc2]b

RusA HjcT4 endoVII

T7 endoI

aProteins in parenthesis form a complex. bProteins in brackets are a homolog or alternative name of the protein

Figure 1 Schematic diagram of the nuclease activity. The twostrands of DNA are schematically drawn. The cleavage madeby the nuclease is represented by arrowhead

Structure and function of DNA repair nucleaseT Nishino and K Morikawa

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Base excision repair

Abasic sites within DNA duplexes are frequentlyproduced by the actions of various DNA glycosylasesinvolved in the base excision repair pathway, in

addition to the spontaneous hydrolysis of bases. Theseapyrimidine or apurine (AP) sites are removed by APendonucleases which cleave the phosphdiester bondnext to an abasic site (Figure 2) (reviewed in Mol etal., 2000a). E. colicells contain two AP endonucleases:endonuclease IV (endoIV) and exonuclease III(exoIII). Interestingly, these two enzymes show nosequence similarity to each other; although their APendonuclease activities are quite similar. In eukaryotes,there seems to be a single, major AP endonucleaseworking in each organism. APN1, the yeast homologof E. coli endoIV, shows sequence and catalyticactivity similarity to endoIV. The absence of APN1results in enhanced sensitivity to oxidative damage and

alkylating agents (Ramotar et al., 1991). Mammalianorganisms, including humans, bear Ape1, which sharessequence similarity with E. coli exoIII but lacks theintrinsic 3 to 5 exonuclease activity. In addition tothe AP endonuclease activity, Ape1 also plays a majorrole in sensing the redox state of the cell (Xanthou-dakis et al ., 1992). The loss of Ape1 generatesembryonic lethality in mice (Wilson and Thompson,1997).

Mismatch repair

In prokaryotes, mismatch repair is conducted mainly

by the MutSLH proteins, while the Vsr protein is

responsible for mismatches in certain sequences(reviewed in Modrich and Lahue, 1996; Yang, 2000;Tsutakawa and Morikawa, 2001). In the MutSLHsystem, the MutS protein recognizes and bindsmismatched base moieties of DNA. MutL mediates

the interaction between the MutS and MutH proteins.MutH recognizes a hemimethylated GATC sequence,and cleaves next to the G of the non-methylated strand(Figure 2). The cleavage activity of MutH is enhancedby the MutL protein, although its mechanism remainsunclear. Vsr is a mismatch-specific endonucleaseinvolved in very short patch repair, and recognizes aTG mismatch at the specific sequence CT(A/T)GG,where the mismatch occurs at the second thymine,upon spontaneous deamination. Vsr makes an incisionnext to the mismatched base. In both cases, after thenick has been introduced, these sites are degraded bythe RecJ, ExoVII, or ExoI nuclease and are resynthe-sized by the DNA polymerase.

Nucleotide excision repair

Nucleotide excision repair (NER) is primarily used toprocess DNA damage that is not repaired by baseexcision repair. These forms of damage involve thosegenerated by the UV radiation and the large adductsproduced by various chemicals. In the NER pathway, ashort stretch of DNA containing the damagednucleotide is removed. During this process, twoincisions, on the 5 side and the 3 side are made bytwo different nuclease reactions (Figure 2) (reviewed inPetit and Sancar, 1999; Prakash and Prakash, 2000; de

Boer and Hoeijmakers, 2000). In bacteria, this dual

Base Excision Repair

Figure 2 Nuclease associated DNA repair pathways. The substrate DNAs are drawn schematically and the arrowheads denotenuclease cleavage. RNA regions are drawn in bold line


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incision is performed by the UvrB-UvrC complex. Inbudding yeast, Rad2 and the Rad1-Rad10 complexmake the 5 and 3 incisions, respectively. The sameprocess in mammalian cells is conducted by theirhomologs, XPG and XPF-ERCC1, respectively. Dele-tions or mutations introduced into these nucleases

cause sensitivity to UV damage, and result in cancerformation. In addition, abnormalities of these proteinscause defects in neural development.

Double strand break repair

Double strand breaks are generated by the accidentalhalt of fork progression during replication or byionizing radiation and strand incision chemicals. Theyare also generated as an intermediate state duringmeiosis and V(D)J recombination. These double strandbreaks are repaired through the two main pathways ofnon-homologous end joining and homologous recom-bination. In either case, the ends of the double strand

breaks must be processed to initiate the repair reaction(Figure 2). Mre11 is a multi-functional nucleaseinvolved in the processing of the DNA ends orhairpin structures (reviewed in DAmours and Jack-son, 2002). While Mre11 itself exhibits a ssDNAexonuclease activity, its complex with Rad50 processesdouble strand break ends. Moreover, in the presenceof ATP, Rad50 activates the cleavage activity ofMre11. Mutations introduced into Mre11 cause anataxia-telangiectasia-like disorder (Stewart et al .,1999).

V(D)J recombination involves a reaction process, inwhich hairpin DNAs are opened, and subsequently,

both ends are connected. Recently, the Artemis : DNA-PK complex was shown to participate in this openingreaction (Ma et al., 2002). Although Artemis alonepossesses a ssDNA exonuclease activity, its complexformation with DNA-PK allows the processing of thedouble strand break ends to open the hairpin structure.Defects in each protein cause severe immunodeficiency(Blunt et al., 1995; Kirchgessner et al., 1995; Moshouset al., 2001).

In homologous recombination, two homologousDNA strands are paired and are connected by D-loopstructures or Holliday junction intermediates. Inbacteria, the RuvC protein cleaves the Hollidayjunction at two symmetrical sites near the junction

center to resolve the junction into two dsDNAs (Figure2). Similar junction resolving enzymes have also beenfound in other bacteria, bacteriophages, and archaea(reviewed in Sharples, 2001). In eukaryotes, FEN1,XPF-ERCC1, and Mus81 are known to cleave the D-loop structure, while Cce1/Ydc2 processes Hollidayjunctions in mitochondria.

Structural classification of DNA repair nucleases

The primary sequences of nucleases are often poorlyconserved, except for the motifs related to catalytic

sites. The functional and biochemical properties of

many nucleases have been studied extensively.However, in some cases, it is very difficult to identifythe actual functional targets of the nucleases, becauseof their broad substrate specificity. Nevertheless, manycandidates for nucleases are available from variousgenome sequences, and their functional properties can

be inferred by sequence comparisons with other well-studied nucleases. For instance, Koonin and hisassociates have successfully classified nucleases, phos-phoesterases, and phosphatases into several families,based on extensive data base analyses of the primarysequences (Aravind and Koonin, 1998a,b; Aravind etal., 1999). This classification has also revealed therelationships between nucleases and identified severalnew nuclease families.

In addition to the classifications of primarysequences, 3D structural data have been rapidlyaccumulating with respect to the proteins involved inDNA repair, including nucleases. Most of theirstructures were solved in the DNA-free states, although

a number of them were determined in complex withcofactors or/and DNA (Table 2). The classification ofnucleases in terms of their 3D structures provides moredefined properties, since it is accepted that the 3Dstructures are much less diverged and more closelyrelated to the functions than the primary sequences. Asa matter of fact, in the type II restriction endonu-cleases, all of the structures share the common coremotif, which includes the active sites, and thus could begrouped into a single folding family, despite its primarysequence diversity (reviewed in Pingoud and Jeltsch,2001). In the following section, we describe each of thefolding families of the DNA repair nucleases, classifica-

tions based on the SCOP database (Figure 3 and Table3) (Murzin et al., 1995).

RNaseH-like fold

The RNaseH-like fold, which is one of the mostubiquitous architectures in the protein world, has beenfound in RuvC, RNaseH, integrase, transposase, andproofreading exonucleases (Figure 3a). The corestructure contains a five-stranded b-sheet flanked byseveral a-helices. The strand order is 32145, with strand2 anti-parallel to the others. The active site residues,which are constituted according to the DDE motif, arelocated on one side of the sheet. These three (or

sometimes four) acidic residues coordinate the metals,which are essential for the catalytic reaction. Forinstance, the crystal structures of RNaseHI exhibit one(Katayanagi et al ., 1993) or two (Goedken andMarqusee, 2001) metals bound to the active site.Similarly, the active site of the proofreading subunitof DNA polymerase III coordinates two metals(Hamdan et al., 2002). The cocrystal structure withTMP revealed that the phosphate moiety is directlycoordinated between the two metals, as it mimics theproduct DNA. A similar structure is also observed inthe DNA complexes of the Klenow fragment ofpolymerase I (Beese and Steitz, 1991) and the RB69

DNA polymerase (Shamoo and Steitz, 1999).


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Resolvase-like fold

This fold has been found in gd resolvase, 5 3exonucleases, and FEN1 (Figure 3b). It is similar to

the RNaseH-like fold, with a five-stranded b-sheet.However, it possesses a different strand order, which isdefined as 21345 with strand 5 anti-parallel to theothers. FEN1 possesses two acidic clusters formed byfour or three conserved aspartate/glutamate residues.These clusters each coordinate a metal, and areseparated by 5 A from each other (Hwang et al.,1998; Hosfield et al., 1998).

Restriction endonuclease-like fold

The structures of restriction endonucleases revealedthat their catalytic domains share common fold

architecture (Figure 3c). The core fold comprises a

five-stranded b-sheet flanked by several a-helices. Thestrand order is 12345, with strand 2, and in some cases,strand 5, anti-parallel to the others. A conservedPDXn(D/E)XK sequence is located on one side ofthe b-sheet, and is involved in the formation of thecatalytic centers in most restriction endonucleases.

Similar sequences are also found in several DNArepair nucleases, such as MutH, Hjc, and T7 endoI,which are categorized into essentially the same foldingfamily. The Vsr endonuclease also shares a similar fold,whereas the (D/E)XK sequence is replaced by FXH,where histidine participates in catalysis (Tsutakawa etal., 1999b). The active sites in endonucleases with therestriction endonuclease-like fold coordinate up tothree metals depending upon the enzyme.

RecJ-like fold

This fold was recently identified by the determinationof the RecJ nuclease structure (Yamagata et al., 2002)

(Figure 3d). Previous sequence analyses have shownthat this family includes RecJ and the phosphoes-terases, which contain conserved phosphoesterasemotifs (Aravind and Koonin, 1998a). The structurerevealed a novel fold, which consists of a five-strandedparallel b-sheet flanked by six a-helices. The strandorder of the b-sheet is 21345. On one side of the b-sheet, four phosphoesterase motifs form a cluster,which contains five invariant aspartates and twoconserved histidines. The structure of the crystal,grown in the presence of 100 mM MnCl2, exhibits astrong metal peak coordinating three of the aspartatesand one of the histidines. These residues, which

constitute part of the active site, are likely toparticipate in the cleavage reaction.

Metallo-dependent phosphatase fold

Mre11 and several phosphatases, including the purpleacid phosphatase and the ser/thr phosphatases, sharethis fold (Figure 3e). The core structure contains twob-sheets, which are sandwiched by a-helices to form afour-layered structure. The primary sequence of thisfamily contains the conserved phosphoesterase motifsusually constituted by six histidines, three aspartates,and an asparagine, which form a cluster on one side ofthe b-sheet. The cocrystal structure of Mre11 with Mn

and dAMP shows two manganese ions bound to theactive site, and these two metals are simultaneouslycoordinated to the phosphate moiety, thus mimickingthe product-bound state (Hopfner et al., 2001). Theactive sites of the ser/thr phosphatases bind two metals(zinc and iron) with a similar coordination scheme(Griffith et al., 1995).

DNaseI-like fold

This fold is found in DNaseI, ExoIII, and Ape1(Figure 3F). It is also observed in some phosphatases,such as inositol 5-phosphatase. These nucleases share a

four-layered structure containing an a/b sandwich, as

Table 2 Structural analysis of DNA repair proteins

Free/partial +cofactor +DNA

Replication coupled repair proteinsReplicational polymerase * * *(+proofreading domain)a

Repair polymerase * * *

Error prone/free polymerase * * *PCNA * *FEN1a * *

Damage ReversalPhotolyase * *Ada/Ogt * *MutT * *

Base excision repairAag Glycosylase * * *AlkA * * *MutM/Fpg/EndoVIII * * *MutY/Ogg/EndoIII * * *UDG * * *TAG * * *ExoIII/Ape1a * * *

EndoIVa

* * *

Mismatch repairMutS * * *MutL/Pms2 *MutHa *Vsra * * *

Nucleotide excision repairUvrB * *

Double strand break repairKu70-80 *Mre11a *Rad50 * *Xrcc4-Ligase IV *

Homologous recombinationRecA/Rad51 * *RecG * * *RecJa * *RuvA * *RuvB * *Holliday junction * *

resolvase (RuvC etc.)a

aContain nuclease activity


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found in the metallo-dependent phosphatases, althoughthe b-sheet topology and the environments around theactive sites are different. The active site is located onone side of the b-sheet, which assembles severalconserved acidic residues. The crystal structures ofDNaseI (Suck et al., 1988) and ExoIII (Mol et al.,1995) revealed a single metal ion bound to the activesite. On the other hand, one (Gorman et al., 1997) ortwo (Beernink et al., 2001) metals were observed in thefree form of Ape1. The Ape1-DNA complex structurerevealed one metal, coordinated with the acidicresidues and the cleaved phosphate in the active site

(Mol et al., 2000b).

TIMb/a barrel foldThe TIM barrel was first observed in triosephosphateisomerase, and is now known to be the mostubiquitous fold adopted by various enzymes withdiverse functions (Farber and Petsko, 1990) (Figure3g). It forms the a8/b8 barrel structure, where a barrel-like parallel b-sheet is surrounded by eight a-helices. Inthis fold, the key residues for the enzymatic activity areusually located on the C-terminal side of the barrel.The structure of E. coli endoIV was the first DNArepair enzyme structure with the TIM barrel (Hosfieldet al., 1999). The active site contains a cluster of threezinc ions coordinated by histidines and aspartates. The

endoIV-DNA complex structure revealed how thesezinc ions coordinate the cleaved AP site.

His-Me finger endonuclease fold

T4 endonuclease VII (T4 endoVII) and several othernucleases, such as the colicin nucleases, Serratianuclease and I-PpoI intein, contain this folding motif(Figure 3h). It is usually embedded as a constituent oflarger architectures. The core fold is a b-hairpinflanked by two helices. Within the hairpin, severalhistidines and acidic residues form a cluster andcoordinate a catalytically important divalent metal. Inthe case of T4 endoVII, a single metal ion is

coordinated to aspartate, glutamate, and asparagines(Raaijmakers et al., 1999). The I-PpoI-DNA complexstructure revealed that a histidine lies within thedistance of hydrogen-bond from the scissile phosphategroup in the metal-containing active site (Galburt etal., 1999).

DNA recognition by DNA repair nuclease

The binding modes of DNA nucleases are roughlydivided into two categories, corresponding to non-specific and specific associations. Both modes are

important for efficient and accurate recognition

Figure 3 Folding patterns of DNA repair nucleases. The core folding is drawn schematically. The yellow arrows indicate the coreb-sheet, where the strand orders are numbered on the top. a-helices are shown as blue cylinders. The positions of the bound metalsare marked by black circles. Representative repair nuclease of the folding is written in parenthesis

Table 3 Structural classification of DNA repair nucleasesRNaseH-like fold

RNaseHRuvCExoIproofreading (exonuclease domain)

Resolvase-like foldFEN1

Restriction endonuclease foldMutHVsrT7 endoIHjc

RecJ foldRecJ

Metallophosphatase foldMre11

DNaseI foldExoIII/Ape1

TIMb/a barrel foldEndoIV

His-Me finger nuclease foldT4 endoVII


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between enzymes and DNA. Non-specific DNAbinding allows enzymes to scan for target sequencesor damage by a rapid diffusion process along theDNA. Once the nuclease finds its proper target, specificinteractions are made to dock the active site residuescorrectly to the chemical groups within the DNA for

cleavage. These two binding modes have beenvisualized within the crystal structures of the type IIrestriction endonucleases (reviewed in Pingoud andJeltsch, 2001). In the cases of EcoRV, BamHI, andPvuII, the non-specific binding involves a weakassociation, which is contributed by an electrostaticinteraction between the minimum surface area of theprotein and the DNA, and the overall shape of theDNA remains in the canonical B-form, without seriousdeformations. By contrast, in the specific complex, theDNA is buried within the deep cleft of the protein in asequence-specific manner, accompanied by the remark-able deformation of the DNA duplex, which isrequired for the cleavage by the enzyme.

This scheme can be generally applied to DNA repairnucleases as well. The nuclease surfaces are rich inbasic residues, which form positive surfaces competentfor electrostatic interactions with DNA. Somenucleases, such as MutH or Vsr, which both sharethe restriction endonuclease fold, possess partialcompetences for sequence-specific recognition, just likerestriction endonucleases. However, most DNA repairnucleases recognize certain mismatches, forms ofdamages, or particular backbone structures of DNA.Therefore, they require additional and unique bindingmechanisms for specific interactions with DNA.Although the information available for DNA repair

nuclease-DNA complexes is limited, they can stillprovide considerable insights into such recognitionmechanisms.

Base flipping out

Base flipping out has been observed in many DNAglycosylases and methyltransferases (reviewed inRoberts and Cheng, 1998; Vassylyev and Morikawa,1997; Mol et al., 2000a; Parikh et al., 2000). Theflipping-out of a base is defined as the localconformational change of a DNA duplex, where abase is swung out from inside of the helix into anextrahelical position and is usually inserted into the

binding pocket of the protein. The space created bythis process of base pair disruption is occupied byprotein atoms, which are often involved in catalyticreactions. This mechanism is observed in the twocrystal structures of the AP endonucleases, Ape1 andEndoIV (Figure 4a,b), which were both complexed withDNA duplexes containing an AP site in the middle.These two structures showed a similar base flipping,but different fitting modes, between the DNA and theproteins.

In the Ape1-DNA complex, the abasic nucleotidewas flipped out into the enzyme pocket (Mol et al.,2000b) (Figure 4a). The gap was filled on the minor

groove side by two methionines (Met270, Met271) and

on the major groove side by arginine (Arg177). Theseinsertions generate a sharp kink of the DNA duplex atthe abasic site. The comparison of the free form withthe complex revealed a small difference, suggesting thatthe surface of the enzyme contains a preformed pocketto be filled by the flipped out base. Thus, it is likely

that Ape1 searches its target by scanning for a possiblebase flipping site. Once Ape1 finds the target, the baseflips out into the enzyme pocket, and the remaininggap is occupied by the inserted arginine to stabilize theprotein-DNA complex. Biochemical experimentsconfirmed the role of this arginine, which whenmutated to alanine, resulted in elevated enzymeturnover. (Mol et al., 2000b).

In the endoIV-DNA complex, an abasic site issimilarly flipped out into the protein pocket (Hosfieldet al., 1999) (Figure 4b). However, the conformation ofthe DNA duplex is drastically different from that ofApe1-DNA. The orphan base opposite the abasic sitealso occupies an extrahelical position. Consequently,

the DNA duplex is sharply bent (908) at the abasic site.The gap made by both flipped out nucleotides is filledby arginine (Arg37), tyrosine (Tyr72), and leucine(Leu73) inserted from the minor groove. In contrast tothe preformed pocket of Ape1, the recognition loops ofendoIV undergo a drastic conformational change uponDNA binding. The residues involved in base flippingare located in this loop. It is likely that endoIV scansthe DNA duplex on the minor groove side by thisDNA recognition loop. Once the enzyme finds thetarget, it inserts all of the DNA-penetrating residues,and flips the two bases into extrahelical positions.

Insertion of aromatic side chains

Another important factor in the recognition betweenrepair enzymes and DNA is the insertion of aromaticamino acids into DNA duplexes. This is different fromthe insertion of amino acid side chains, which fill upthe gap created by a base-flipping out. A representativecase was observed in the Vsr-DNA complex (Tsutaka-wa et al., 1999a) (Figure 4c). Vsr recognizes a TGwobble mismatch base pair located in a five base pairlong recognition sequence. In the close vicinity of themismatch, Vsr intercalates three conserved aromaticamino acids (Phe67, Trp68, Trp86) from the majorgroove. In addition to the inserted helix from the

minor groove, this insertion expands the space betweenthe TG mismatch and the adjacent base pair, while thebase pair itself is not disrupted. A similar insertion ofaromatic residues was observed in the MutS-DNAcomplex, where the aromatic side chain of a conservedphenylalanine was inserted next to a mismatched orgapped base pair (Lamerset al., 2000; Obmolova et al.,2000).

The exonuclease domain of DNA polymerase usesaromatic residues for the correct positioning of thenucleotides (Figure 4d). In the editing complex ofRB69 DNA polymerase with its substrate DNA, twosingle-stranded nucleotides are located in the groove of

the exonuclease domain (Shamoo and Steitz, 1999).


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One of the nucleotides is held by forming a hydrogenbond with the side chain of Arg260. Anothernucleotide, whose backbone is cleaved, is located moredeeply within the exonuclease pocket, and is segregated

from the remaining region by the insertion of twoaromatic side chains (Phe123 and Phe221) to separatethe two nucleotides. These phenylalanines create a wall,and thus the base is correctly positioned within theactive site pocket.

In vivo and in vitro experiments, measuring the UVsensitivity and probing with potassium permanganate,have demonstrated that in E. coliRuvC, the aromaticside chain of Phe69 plays a crucial role in specificrecognition with the Holliday junction (Yoshikawa etal., 2001). Phe69 lies in the protruding loop and directsits side chain into the catalytic cleft, which accom-modates one of the DNA duplexes. A similar residue isalso present in the yeast structural homolog, Ydc2,

whereas it is absent in another yeast homolog, Cce1.Consequently, the detailed structural view of recogni-tion mechanism between RuvC and the junction DNAis required to solve the complex directly.

Active site environments of DNA repair nucleases

All nucleases cleave the same phosphodiester bond, toleave 5-phosphate and 3-OH groups at the producedsegments. Similar reactions are conducted by phospha-tases and ribozymes, although their catalyticmechanisms have not been clarified yet. The overall

aspect of this enzymatic scheme is that the attacking

water is activated by a general base in the nucleaseactive center, which usually bears a metal cofactor.This activation is performed by protein side chains ordivalent metals. The activated water is converted to a

hydroxide, which attacks the phosphate, thus formingthe transition state intermediate. There are two modesfor this nucleophilic substitution: associative anddissociative. The associative mechanism involves theformation of a pentacovalent intermediate with ahydroxide, followed by the release of a leaving group.In this mechanism, a general base is required togenerate the hydroxide, and a general acid is needed tostabilize the leaving group. The dissociative mechan-ism, on the other hand, does not require this generalacid and general base, and they form a metaphosphateintermediate, which requires more stabilization of thetransition intermediate. Many nucleases are assumed tofollow the associative mechanism, while alkaline

phosphatase uses the dissociative mechanism.A large number of nucleases utilize metal cofactors

for the hydrolytic reaction. They are proposed to playany one or a combination of the following roles(Figure 5) (Jencks, 1969): (1) positioning the substrateand/or the attacking nucleophile; (2) enhancing thenucleophilicity of the phosphate at the scissile bond; (3)activating the nucleophile; (4) neutralizing the negativecharge in the transition state; (5) facilitating thedeparture of the leaving group. To examine theseroles, various metals are recruited to the nucleaseactive sites. While the utilized metal may differ,depending upon the nuclease, magnesium or manga-

nese is the most common metal for catalysis, and in

Figure 4 DNA recognition by DNA repair nuclease. Ribbon diagram of the DNA repair nuclease. (Left panel) Overall structure ofthe protein-DNA complex. (Right panel) Close-up view of the boxed region. Proteins are represented by a yellow ribbon diagram,and the side chains involved in DNA recognition are displayed and numbered with a stick model. The bound DNA is shown as awhite stick model, and the flipped out nucleotides are colored red. The observed metals are shown as spheres. Blue, zinc; light blue,manganese; red, magnesium; gray, calcium. (a) endoIV (b) Ape1 (c) Vsr (d) RB69 polymerase exonuclease domain


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rare cases, zinc is used. The magnesium ion appears tobe transiently recruited to the active sites, whereas zinc

and manganese are more tightly bound to the catalyticcenters.

EndoIV contains three zincs, which are coordinatedby five histidines, two glutamates, and two aspartates,in addition to two water molecules (Figure 6a). Thesemetals are so tightly coordinated to the enzyme thateven EDTA cannot chelate them (Levin et al., 1991).Two of the three zinc atoms are likely to be involved ingenerating the attacking nucleophile, in cooperationwith the carboxyl side chain of Glu261. Furthermore,all three of the metals coordinate the phosphate moietyafter cleavage (Hosfield et al., 1999).

Mre11 coordinates two manganese ions through five

histidines, two aspartates, and one asparagine (Figure6b) (Hopfner et al., 2001). The two manganese ionsdirectly coordinate the phosphate moiety of the dAMP.When magnesium is substituted for manganese, theycan only occupy one of the two metal binding sites,and the nuclease is inactive. This indicates that bothmetals are required for the nuclease activity.

As for the nucleases that require magnesium cationsfor catalyis, the number of metals and their positionsin the active sites are more ambiguous. They arecoordinated with protein atoms in a more transientmanner. This relatively weak binding, and the fact thatthe electron number of the magnesium cation iscomparable to a water molecule, make the clear

identification of the metal positions more difficult. Inaddition, the number of bound metals may change,depending upon different crystallization conditions.The free form structure of the Ape1 crystal, obtainedunder acidic conditions, revealed a single, bound metal(Samarium) (Gorman et al., 1997), whereas the crystalobtained at a neutral pH contained two metals (Lead)in the active site (Beernink et al., 2001). These Ape1data indicate that the metals occupy multiple sites,which are affected by the protonation of the acidicresidues. It appears that two metals are required forcatalysis, since Ape1 is only active at a neutral pH.However, the actual numbers and the role of each

metal cannot be clarified at the moment, because the

structure of the Ape1-DNA complex was obtainedunder acidic conditions, and only one manganese ion is

bound to the product DNA cleaved at the abasic site(Figure 6c) (Mol et al., 2000b). Similar ambiguity withrespect to the number of metals was reported forRNaseHI, such as one magnesium (Katayanagi et al.,1993) and two manganeses (Goedken and Marqusee,2001). In the Vsr-DNA complex structure, twomagnesium ions are clearly observed in the active site,with one of the metals holding both the 5 phosphateand 3 OH groups (Figure 6d, Tsutakawa et al., 1999a).Two metals are also found in the exonuclease domainof polymerases (Calcium) (Figure 6e) (Shamoo andSteitz, 1999) and in T7 endoI (Manganese) (Hadden etal., 2002), although the two sites are not equivalent

between the two enzymes, and one of the two metalsshows partial occupancy.

Future perspectives

With the rapid accumulation of metal binding siteinformation, various catalytic mechanisms have beenproposed, including the classical two metal bindingmechanism (Beese and Steitz, 1991). However, itappears to us that the actual numbers and positionsof the metals involved in catalysis are too broadlyvaried from enzyme to enzyme to describe theirhydrolytic mechanisms by a unified catalytic scheme.

More detailed structural information, hopefullycombined with biochemical data, is essential to obtainclear insights into the metal dependent nucleasemechanisms. Meanwhile, the large diversity in nucleasearchitectures suggests that they can specifically recog-nize DNA substrates by virtue of the large variety ofsurface properties, which were adopted throughselection over an extremely long period. In particular,the nucleases involved in DNA repair have acquired aspecial damage recognition system. At present, much ofthe structural information is based on that ofprokaryotic and archaeal proteins. Eukaryoticnucleases obviously hold more complicated structures

and properties, because they must bear eukaryote-

Figure 5 Schematic diagram of cleavage by DNA repair nucleases. X, Y, Z-H denote general base, Lewis acid, and general acid,respectively. Numbers in circles indicate reaction steps where the metal cofactors may be involved


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specific regulatory mechanisms involving protein protein and protein-DNA interactions. Further 3Dstructural characterizations of the eukaryotic DNArepair nucleases should provide additional variations orconserved architectures of protein folding, whilestructural analyses of their complexes with DNAsubstrates will clarify the recognition mechanisms.

AcknowledgementsWe regret that the limit of space may have not allowed usto site all works in the field. We thank Kayoko Komori forcritical reading of the manuscript and helpful comments. TNishino is a research fellow of the Japan society for thepromotion of sciences. This research was partly supportedb y NEDO (New En ergy and Ind ustrial Tech no lo gyDevelopment Organization).

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Structure and Function of DNA Repair Nuclease