DNA Mismatch Repair and Genetic Instability

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September 29, 2000 11:40 Annual Reviews AR116-13

Annu. Rev. Genet. 2000. 34:359–99Copyright c© 2000 by Annual Reviews. All rights reserved

DNA MISMATCH REPAIR AND GENETIC

INSTABILITY

Brian D Harfe and Sue Jinks-RobertsonDepartment of Biology, Emory University, Atlanta, Georgia 30322;e-mail: [email protected]

Key Words mutation, mutator, recombination, DNA damage, cancer

■ Abstract Mismatch repair (MMR) systems play a central role in promotinggenetic stability by repairing DNA replication errors, inhibiting recombination be-tween non-identical DNA sequences and participating in responses to DNA damage.The discovery of a link between human cancer and MMR defects has led to an explosionof research on eukaryotic MMR. The key proteins in MMR are highly conserved frombacteria to mammals, and this conservation has been critical for defining the compo-nents of eukaryotic MMR systems. In eukaryotes, there are multiple homologs of thekey bacterial MutS and MutL MMR proteins, and these homologs form heterodimersthat have discrete roles in MMR-related processes. This review describes the geneticand biochemical approaches used to study MMR, and summarizes the diverse rolesthat MMR proteins play in maintaining genetic stability.

CONTENTS

INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 360MISMATCH REPAIR PROTEINS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 360

The Bacterial Paradigm. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 360Mismatch Repair Proteins in Eukaryotes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364

MISMATCH REPAIR IN MUTATION AVOIDANCE . . . . . . . . . . . . . . . . . . . . . . 371Mutation Avoidance in Bacteria. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 371Mutation Avoidance in Yeast. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 372Mutation Avoidance in Mammals. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 375

ROLES OF MMR IN RECOMBINATION PROCESSES. . . . . . . . . . . . . . . . . . . . 377Mismatch Correction in Recombination Intermediates. . . . . . . . . . . . . . . . . . . . . 378Regulation of Recombination Between Nonidentical Sequences

(Anti-Recombination). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 378Nonhomologous Tail Removal. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 381

ROLES OF MMR PROTEINS IN DNA DAMAGE-RELATED PROCESSES. . . . . 382MEIOTIC-SPECIFIC ROLES OF MMR PROTEINS. . . . . . . . . . . . . . . . . . . . . . . 384SUMMARY AND FUTURE PERSPECTIVES. . . . . . . . . . . . . . . . . . . . . . . . . . . 385

0066-4197/00/1215-0359$14.00 359

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360 HARFE ¥ JINKS-ROBERTSON

INTRODUCTION

The mismatch repair (MMR) system is critical for maintaining the overallintegrity of the genetic material, and the basic features of this system have beenhighly conserved during evolution. The MMR system is best known for its role inthe post-replicative repair of DNA polymerization errors, which is critical for keep-ing mutation rates at an acceptably low level. In addition to recognizing replication-generated mismatches, MMR proteins also recognize mismatches in heteroduplexrecombination intermediates. Mismatch recognition in recombination intermedi-ates can elicit a repair process that leads to a genetically detectable gene conversionevent, or can trigger an anti-recombination activity that prevents the recombina-tion event from going to completion. The anti-recombination activity of MMRproteins promotes genome stability by inhibiting interactions between divergedsequences present in a single genome or derived from different organisms. Inaddition to recognizing and processing DNA mismatches comprised of “normal”bases, the MMR system also plays a role in responses to DNA damage. Finally,eukaryotic MMR proteins have recombination roles that are unrelated to mismatchrecognition, and some proteins have evolved essential roles in meiotic chromo-some metabolism. This review summarizes the diverse roles of MMR proteins inpromoting genetic stability and focuses on genetic consequences of MMR defects.

MISMATCH REPAIR PROTEINS

Proteins unique to the MMR system (“Mut” proteins) were originally identifiedin prokaryotic organisms, where their loss enhances the accumulation of DNAreplication errors and results in a mutator phenotype. The first step in the cor-rection of replication errors via the MMR system involves efficient recognitionof helical distortions (mismatches) resulting from nucleotide misincorporation orDNA polymerase slippage. Next, the newly synthesized DNA strand contain-ing the incorrect information must be selectively removed and re-synthesized.Strand discrimination is an essential feature of all MMR systems; in its absence, areplication error is just as likely to be used as a template for repair as it is to be re-paired. Whereas the latter steps in MMR require proteins involved in general DNAmetabolic processes, the initial mismatch recognition and removal steps requirespecialized Mut proteins, which are highly conserved evolutionarily. Below, theproteins and mechanism of the prototypeEscherichia coliMMR system are de-scribed, as well as corresponding MMR components that have been identified froma wide variety of eukaryotic organisms. These proteins are summarized in Table 1.

The Bacterial Paradigm

The E. coli MMR system has been completely reconstituted in vitro [reviewedin (157)] and involves three dedicated proteins: MutS, MutL, and MutH. MutS

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MISMATCH REPAIR 361T

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is an ATPase that effects mismatch recognition, MutL is an ATPase that cou-ples mismatch recognition by MutS to downstream processing steps, and MutHis a methylation-sensitive endonuclease that targets repair to the newly synthe-sized DNA strand (Figure 1, see color insert). The MutS protein binds as a ho-modimer to DNA and shows in vitro specificity for base-base mispairs and forinsertion/deletion loops up to 4 nucleotides (nt) in length (170). Deletion analysisof MutS indicates that the C-terminal end of the protein is involved in dimer-ization, while the N-terminal end is important for binding mismatch-containingDNA (235). A P-loop motif for nucleoside triphosphate binding is located in theC-terminal half of MutS, and ATP binding/hydrolysis promotes dissociation ofthe MutS-mismatch complex, a step that appears to be essential for the down-stream steps in MMR. Mutation of the conserved ATP-binding domain of MutSis associated with a dominant negative mutator phenotype in vivo (234), pre-sumably because mismatches are bound in an irreversible manner that precludesbinding of repair-competent complexes. As visualized by electron microscopy,addition of ATP to a mismatch-bound MutS complex triggers the formation of anα-shaped loop structure with MutS at the base of the loop and the mismatch at theapex (10). Modrich and colleagues have proposed that this structure is generatedby ATP hydrolysis-dependent bidirectional translocation of MutS away from themismatch (157).

The crystal structures of theE. coli MutS dimer (T Sixma, personal communi-cation) and theT. aquaticusMutS dimer (W Yang & P Hsieh, personal communi-cation), each complexed with a mismatch-containing oligonucleotide, have beensolved. The structures are very similar and provide invaluable insight into proteinfunction. Although both proteins function as homodimers, the monomer subunitshave different conformations. The formation of a structural heterodimer from iden-tical protein subunits is particularly significant from an evolutionary perspectiveas it explains why the eukaryotic MutS complexes exist only as heterodimers(see below). The crystal structure reveals that MutS has two large channels, one ofwhich constitutes the mismatch binding site and contains the conserved phenylala-nine identified in the earlier crosslinking studies (145). Only one subunit actuallybinds the mismatch, although both contact the DNA, forming a clamp. The crys-tal structure confirms that the highly conserved helix-turn-helix domain at theC terminus is important for dimerization of MutS, and demonstrates that both theATPase site and the DNA binding site are composite sites utilizing domains fromboth subunits. Consistent with the composite nature of these sites, disruption ofthe helix-turn-helix domain results in concomitant losses of dimerization, ATPhydrolysis, and mismatch binding (P Hsieh & W Yang, personal communication).

The strand discrimination signal in theE. coli MMR system is provided bythe transiently unmethylated state of newly synthesized DNA [reviewed in (157)].Specifically, the MutH protein cleaves the unmethylated strand of a hemimethy-lated GATCdammethylation site, thereby marking the nicked strand for exonu-cleolytic removal and resynthesis. Incision is mismatch dependent, with the MutHendonuclease activity being activated in vitro by a complex of MutS, MutL, and

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MISMATCH REPAIR 363

mismatched DNA (12). MutH can nick DNA on either side of a mismatch and,depending on which side cleavage occurs, either a single-strand specific 5′ to 3′

or a 3′ to 5′ exonuclease degrades the mismatch-containing nicked strand. In theE. coli system, there are two 5′ to 3′ exonucleases (exonuclease VII and the RecJexonuclease) and two 3′ to 5′ exonucleases (exonuclease I and exonuclease X)that can effect removal of the nicked strand (38; S Lovett, personal communi-cation). Because the exonucleases are single-strand specific, the activity of theUvrD (MutU) helicase is needed to unwind the duplex molecule. Unwinding bythe UvrD helicase begins at the nick and proceeds in a directional manner towardthe mismatch (46). The methyl-directed nature of theE. coli MMR system is anefficient way to discriminate template and daughter strands during DNA replica-tion, but this mechanism is not universal in prokaryotes (see 37), and is apparentlynot applicable to eukaryotes (see below). In the absence of MutH activity, a pre-existing nick on one strand of a duplex is sufficient to confer strand-specific repairin vitro (131).

Genetic studies have shown that MutL is essential for MMR, but unlike theMutS and MutH proteins, defining its precise role in MMR has been difficult. TheN-terminal region of the MutL family of proteins is highly conserved and containsthe ATP binding/hydrolysis domains (17), while the dimerization domain of MutLappears to reside in the C-terminal region of the protein (57). Crystallographic stud-ies have demonstrated that ATP binding leads to dimerization of the N-terminalportion of the protein as well (16), and the accompanying structural changes arespeculated to play key roles in coordinating the initial steps of mismatch recogni-tion with downstream processing steps. MutL thus would be expected to interactwith multiple MMR components. A direct interaction between MutL and MutShas been demonstrated using affinity purification, and deletion analysis of MutSindicates that the C-terminal region is important for the MutL-MutS interaction(235). In addition to the MutL-MutS interaction, direct interaction between theC-terminal region of MutL and MutH has been demonstrated and MutL alone hasbeen shown to stimulate the endonuclease activity of MutH (96). The C-terminalregion of MutL also directly interacts with UvrD (97) and is involved in activatingthe helicase activity of this protein (237). MutL is speculated to load the UvrDhelicase onto the nick in a directional manner so that DNA unwinding proceedstoward the mismatch. A general model that emerges from the biochemical studiesis that MutL coordinates the mismatch binding activity of MutS with the MutHcleavage and UvrD helicase activities, and thereby directs the strand removalprocess.

In addition to the MutHLS system, which is sometimes referred to as the “longpatch” MMR system,E. coli possesses a second “short patch” MMR system thatshares some components with the MutHLS system [reviewed in (85)]. The VSP(very short patch) system specifically repairs G/T mismatches in the context ofDcm methylation sites to G/C, and its primary role is thought to be in the correctionof mismatches resulting from deamination of 5-methylcytosine to thymine. Thekey protein in the VSP system is the Vsr strand-specific endonuclease, and the

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activity of this pathway is dependent on DNA polymerase I, MutS, and MutL.Consistent with a role for MutS and MutL in the VSP system, overproduction ofVsr disrupts function of the long patch repair pathway (56) and MutL stimulatesbinding of Vsr to a G/T mismatch (57).

Mismatch Repair Proteins in Eukaryotes

In E. coli, MMR is effected by a single MutS protein and single MutL protein,both of which are active as homodimers. All eukaryotic organisms characterizedto date possess multiple MutS homologs (MSH proteins) and multiple MutLhomologs (MLH proteins), with the active forms being heterodimers composedof two different MSH proteins or two different MLH proteins. The human MED1protein has been speculated to be a MutH functional homolog because it interactswith an MLH protein and displays endonuclease activity (21). MED1, however,appears to be closely related to DNA glycosylases/lyases, and it has greater affinityfor fully methylated than hemimethylated DNA. Although no convincing MutHhomologs have been identified in eukaryotes and eukaryotic MMR is not thought tobe methyl directed, it can, as inE. coli, be nick directed (70, 110, 211). It has beenspeculated that the nicks at the 5′ and 3′ ends of Okazaki fragments could providethe strand discrimination signal during lagging strand synthesis. Distinguishingthe leading strand from its template is more problematic, although the growing 3′

end could provide an appropriate strand discrimination signal. Like theE. coliMutS protein, the comparable human complex translocates away from a mismatchin an ATP-dependent manner in vitro (89), and this step may be necessary toengage the machinery that processes the mismatch. Eukaryotic MutS and MutLhomologs interact with proliferating cell nuclear antigen (PCNA; 91, 120, 218),which encircles DNA and tethers DNA polymerase to the template during DNAreplication. It has been speculated that the PCNA interaction might allow the MMRmachinery to use the position of the replication complex to determine which strandshould serve as a template during repair (120), or might trigger involvement of the3′ to 5′ exonuclease activities of DNA polymerases in mismatch removal (89, 216).

Yeast The best-characterized eukaryotic MMR system is that of the yeastSaccharomyces cerevisiae, where there are six MutS (MSH1-MSH6) and fourMutL (MLH1-MLH3 and PMS1) homologs. The diverse functions of the multi-ple MutS and MutL homologs are summarized in Figure 2 (see color insert). MSH1is required for the repair and maintenance of mitochondrial DNA (181); MSH2,MSH3, and MSH6 are required for the stability of nuclear DNA (122, 150); andMSH4 and MSH5 are involved in meiotic recombination processes (109, 182).Based on weak amino acid homology to MutS proteins and an associated anti-mutator activity, it has been suggested that the yeast Hsm3 protein corresponds toa seventh MutS homolog (71). Recent studies, however, have demonstrated that themutator phenotype ofhsm3strains is not exhibited under standard growth condi-tions and only becomes evident when cells are grown very slowly (72, 156). Hsm3p

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MISMATCH REPAIR 365

thus is not involved in repairing replication errors that are removed by the standardMMR machinery, and is not considered here to be a canonical MMR protein.

MSH1 is targeted to the yeast mitochondria via a mitochondrial-targetingsequence (34), and loss of mitochondrial function occurs very rapidly inmsh1strains (181). Consistent with a mitochondrial-specific MMR role, heterozygousMSH1/msh1strains exhibit destabilization of poly(GT) tracts as well as an ele-vated frequency of point mutations in mitochondrial DNA (34, 198). MSH1 is theonly yeast MSH protein that is not functionally dependent on other MutS familymembers, indicating that the active form, like the prokaryotic MutS protein, maybe a homodimer (34). The mitochondrial-specific function of MSH1 suggests thata primitive version of this protein may be the founding MutS family member ineukaryotic organisms, and recent phylogenetic analysis supports this hypothesis(42). According to this model, a primitiveMSH1 gene from a postsymbioticmitochondrion was transferred to the nucleus, followed by loss of the mitochon-drial copy of the gene. During evolution, duplication of the primitiveMSH1genewould have allowed the duplicated copies to evolve new functions, thus giving riseto the diverse MSH proteins present in modern eukaryotic organisms. A predictedMSH1-like protein is encoded by theArabidopsis thalianagenome, but similarhomologs have not been found in other organisms (42). It is interesting to notethat no mitochondrial MutL homolog has been identified in yeast.

Genetic data have demonstrated that yeast MSH2 is required for all mismatchcorrection in nuclear DNA, whereas MSH3 and MSH6 are involved in the repairof distinct subsets of mutational intermediates (see below). The current model ofyeast MMR is that mismatch recognition is effected by two distinct MutS-likeheterodimers composed of MSH2 together with either MSH3 or MSH6. Deletionanalysis of MSH2 indicates that the C-terminal region is important for interac-tion with MSH6 (5), and mutations in the C-terminal helix-turn-helix motif candisrupt interaction with MSH6 (9). In vitro binding studies have demonstratedthat MSH2-MSH6 can bind duplex DNA molecules containing either base-basemismatches or insertion/deletion loops, whereas MSH2-MSH3 binds only to du-plexes containing insertion/deletion loops (5, 93, 115, 151). Both the nature of themismatch and the surrounding sequence context are important determinants ofmismatch binding specificity. Although early studies demonstrated that MSH2alone can also bind mismatches (6), the in vivo relevance, if any, of this obser-vation is unclear. In addition to specific binding to mismatch-containing duplexDNA molecules, binding of MSH2 and the MSH2-MSH6 complex to syntheticHolliday junctions has been reported (7, 152).

The regions of the yeast MSH2-containing complexes that are involveddirectly in mismatch binding have not been well defined, although the specificitypresumably resides in the MSH3 and MSH6 subunits. UV-crosslinking studiesdemonstrating covalent linkage of the MSH6 component of the MSH2-MSH6complex to mismatched DNA support this hypothesis (25, 114). In addition, site-directed alteration of an amino acid suspected to be involved in MSH6 DNA bind-ing abolishes in vitro mismatch binding by the mutant MSH2-MSH6 complex

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(25). Ternary complex formation between MLH1-PMS1 and either mismatch-bound MSH2-MSH3 or MSH2-MSH6 has been observed in vitro (94, 95), and itis assumed that the MSH2-containing heterodimers interact with one of the threeMLH1-containing heterodimers to initiate the yet-to-be-defined downstream stepsin MMR.

Like the MutS protein inE. coli, the yeast MSH proteins possess ATP bindingand hydrolysis activity that is located in the highly conserved C-terminal region.In in vitro mismatch binding assays, addition of ATP to the reaction abolishesMSH2-MSH6 mismatch binding and results in proteolysis-sensitive conforma-tional changes in the complex (9, 95, 206). The ATP-dependent mismatch releasepresumably reflects the translocation of the complex away from the mismatch andoff the ends of the bound DNA fragment (see below and 89). Although MSH2-containing complexes will bind mismatches that are poor substrates for in vivoMMR (e.g. palindromic loops), ATP does not trigger release of such mismatches(5, 151, 228). This indicates the importance of ATP binding/hydrolysis for the com-pletion of the MMR process in vivo. As observed with the bacterial MutS protein,mutant forms of MSH2 or MSH6 that exhibit ATP-insensitive mismatch bindingin vitro behave as dominant negative proteins in vivo (9, 58, 206).

The first MMR protein identified in yeast was the MutL homolog PMS1, whosename reflects the fact thatpms1mutants exhibit increased levels of postmeioticsegregation (129, 227). As postmeiotic segregation results from a failure to repairmismatches in meiotic recombination intermediates,pms1mutants have a corre-sponding decrease in meiotic gene conversion events. The remaining three MutLhomologs (MLH1-MLH3) were identified on the basis of amino acid conserva-tion with MutL (39, 83, 176). MLH1 is the central MutL homolog in yeast, andforms heterodimers with the remaining three MutL homologs (224). The MLH1-PMS1 heterodimer is the major player in MMR (176), with MLH1-MLH2 andMLH1-MLH3 complexes being specialized to repair distinct classes of mutationalintermediates (83, 102). The highest degree of homology between the bacterialMutL protein and the eukaryotic MLH proteins is in the N-terminal region thatcontains the ATPase domains, and site-directed mutagenesis of highly conservedamino acids in this region of either MLH1 or PMS1 generally destroys MMRactivity (167). The interaction domains of MLH1 and PMS1 have been localizedby two-hybrid analysis to the C-terminal region of each protein, and dimerizationhas been shown to be necessary but not sufficient for MMR activity (167).

The remaining members of the yeast MutS family, MSH4 and MSH5, wereidentified because of their effects on meiotic recombination, and neither pro-tein has a detectable role in mismatch correction (109, 182). Consistent with anovel role for these proteins, a conserved N-terminal MutS/MSH domain that hasbeen implicated in mismatch binding is absent in MSH4 and MSH5 (42). MSH4and MSH5 form heterodimers, but neither interacts with MSH2 or MSH6 (174).Similar to the requirement of ATP binding for the MMR activity of the mismatch-binding MSH proteins, mutational alteration of the NTP binding domain of MSH5abolishes its meiotic activity (174).

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The yeast MutS and MutL homologs have been well characterized, but relativelylittle is known about the proteins involved in the processing steps of MMR. Somemutations in the PCNA gene (POL30) result in a strong mutator phenotype (13),and epistasis analysis has suggested that the anti-mutator activity of PCNA worksin the same pathway as that of MSH2 and MLH1-PMS1 (31, 120, 218). As notedpreviously, PCNA interacts physically with yeast MMR proteins, and the involve-ment of PCNA in MMR processes provides a potential link between mismatchrecognition and DNA polymerase-associated strand discrimination and/or pro-cessing. This idea has been strengthened by the observation that the syntheticlethality of apol30-104 rad52double mutant [RAD52is essential for recombina-tion in yeast (168)] can be rescued by anmsh2mutation (31). By analogy with theMutHLS system inE. coli (147), it has been hypothesized that thepol30-104mu-tation may disrupt the strand discrimination process, leading to random nickingof both the template and the nascent strands by the MMR machinery (31). In-discriminate nicking of both strands would produce double-strand breaks that, inthe absence of recombination, would be lethal. The genetic data thus suggest thatmismatch binding may lead to the production of recombination-initiating lesionswhen the presumptive PCNA-generated strand discrimination signal is compro-mised. To date, however, no nicking activity has been associated with eukaryoticMMR systems.

Genetic analyses have implicated the exonuclease activities of four proteins inyeast MMR: EXO1, RAD27 (the yeast FEN1 flap endonuclease homolog), DNApolymeraseδ (Polδ), and DNA Polε (121, 212, 216). Polδ and Polε each possess 3′

to 5′ exonuclease activity that is normally associated with the proofreading functionof DNA polymerases, while EXO1 and RAD27 have 5′ to 3′ exonuclease activity.EXO1 interacts directly with MSH2 (212), so its role in MMR is generally notdebated. Whether RAD27 is indeed involved in MMR is questionable (see 213),and additional experiments are needed to resolve this issue. Finally, the proposedroles of the Polδ and the Polε exonuclease activities in MMR processes are basedon synergistic interactions of alleles encoding exonuclease-deficient polymeraseswith exo1null alleles (216). An involvement of polymerase-associated exonucleaseactivity in MMR raises the intriguing possibility that MMR-associated mismatchremoval may be effected by a “backing up” of the polymerases. Although an in-volvement of polymerase-associated exonuclease activity in MMR is an attractiveidea, recent data demonstrate that an exonuclease-deficient Polδ mutant has a cellcycle progression defect. The data also suggest that the associated mutator pheno-type may be related to activation of an error-prone repair pathway in response to anS-phase checkpoint (A Datta & RD Kolodner, personal communication). Clearly,additional work is needed to clarify what, if any, role DNA polymerase-associatedexonuclease activities have in postreplicative MMR.

Based on analogy with the MMR system inE. coli, one might expect that heli-cases would be involved in eukaryotic MMR. Although none has been identifiedin genetic screens designed to detect MMR defects (216, 236), this could sim-ply reflect functional redundancy of relevant helicases. Alternatively, yeast MMR

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may involve exonucleases that work on double-stranded DNA (e.g. EXO1), andhence there may not be an associated requirement for a DNA unwinding activity.Surprisingly, MSH2 interacts with a large number of the RAD proteins that areinvolved in nucleotide excision repair (22). Genetic data indicate that MSH2 andRAD1 cooperate to repair large loops in recombination intermediates (126) andthat MSH2-MSH3 and RAD1 cooperate to repair large loops in mutation inter-mediates (99). MSH2-MSH3 and RAD1-RAD10 also cooperate to remove thenonhomologous tails generated during recombination processes (207).

In addition to the extensive characterization of the MMR proteins inS. cere-visiae, proteins involved in MMR also have been identified in the fission yeastSchizosaccharomyces pombe. S. pombehomologs of the MSH2 and MSH3 pro-teins inS. cerevisiaeare encoded by theswi4+ andswi8+/msh2+/mut3+ genes,respectively (76, 183). As their genetic names imply, bothS. pombeproteins areimportant for mating-type switching, where they are thought to be involved in cor-rectly terminating DNA copy synthesis (77). Although a direct role of the Swi4protein in MMR has not been demonstrated, loss of the Swi8/Msh2 protein resultsin a mutator phenotype, elevates postmeiotic segregation of genetic markers, anddisrupts chromosome organization during meiosis (183). The only MutL homologidentified to date inS. pombeis encoded by thepms1+ gene, so named because theprotein is most closely related toS. cerevisiaePMS1. Loss of the Pms1 proteinin S. pomberesults in the expected mutator phenotype and elevated postmeioticsegregation (190). Finally, theS. pombegenome encodes a homolog ofS. cere-visiaeexonuclease EXO1, and it should be noted that examination ofS. pombeexo1mutants provided the initial evidence for involvement of this protein in MMR(210).

Studies of meiotic recombination between closely spaced genetic markers haverevealed the existence of a MSH2/PMS1-independent short patch MMR path-way in S. pombe. In contrast to the major long patch MSH2/PMS1-dependentpathway, which recognizes all base-base mismatches except for C/C, the shortpatch pathway efficiently corrects C/C mismatches and plays only a minor rolein the removal of other types of mismatches (78, 191). The minor pathway actsnot only on mismatches present in recombination intermediates, but also pre-sumably removes mismatches that arise during DNA replication (75). Surpris-ingly, the MSH2/PMS1-independent pathway involvesS. pombenucleotide ex-cision repair proteins Rhp14p, Swi10p, and Rad16p, which are homologs of theRAD14, RAD10, and RAD1 proteins inS. cerevisiae, respectively (75). There isno evidence that a comparable MSH2/PMS1-independent MMR pathway exists inS. cerevisiae, and whether such a pathway generally is important in higher eu-karyotes remains to be seen. However, the Drosophilamei-9 gene encodes ahomolog of RAD1 inS. cerevisiae, andmei-9mutants exhibit elevated postmei-otic segregation (194), suggesting the existence of an analogous short patch MMRpathway. Finally, the Uve1p endonuclease inS. pombecleaves duplexes con-taining mismatches in vitro, and may correspond to yet another MMR pathway(125).

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Mammals Mammalian MMR proteins have been the focus of intense researchbecause of their association with human tumor formation [for a comprehensive re-view, see (28)]. With the exception of the mitochondrial protein MSH1, homologsof all other yeast MMR proteins have been identified in mammalian cells. The cur-rent names of the mammalian MutS homologs (MSH2–6) correspond precisely tothe designations given to the yeast proteins, although MSH6 was initially referredto as p160 or GTBP (G/T mismatch binding protein) based on biochemical analy-ses. As in yeast, MSH2 forms heterodimers with either MSH3 or MSH6, and themammalian complexes often are referred to as MutSβ and MutSα, respectively.Interestingly, MutSα is present at much higher levels than MutSβ in culturedcells, and the stabilities of both MSH3 and MSH6 are dependent on the pres-ence of MSH2 (59, 149). Each human MSH protein has two interaction domainswith a similar linear orientation in all three proteins, and the same regions of hu-man MSH2 (hMSH2) interact with both hMSH3 and hMSH6 (92). The mismatchrecognition specificities of MutSα and MutSβ are very similar to those of the cor-responding yeast complexes, with MutSα recognizing base substitution and smallinsertion/deletion mismatches and MutSβ being specific for insertion/deletionmismatches (2, 88, 141, 166). The mammalian MSH4 and MSH5 proteins, liketheir yeast counterparts, appear to be meiosis-specific (24, 65, 169) and also formheterodimeric complexes (24).

The in vitro interaction of the human MSH2/MSH6 complex with mismatch-containing duplexes has been characterized in detail, with a particular emphasis onMSH2/MSH6 structural changes elicited by ADP-ATP exchange. These studieshave been the basis for a MMR model in which the MSH complex functions asa nucleotide-regulated molecular switch (see 73). In this model, the ADP-boundform of the MSH complex is proficient at mismatch binding, and such bindingelicits ADP-ATP exchange. ATP binding is hypothesized to turn the MSH complexinto a hydrolysis-independent “sliding clamp” (89) that leaves the mismatch andslides along the DNA until additional components of the MMR machinery arecontacted. This model is substantially different from that proposed by Modrich andcolleagues, which involves ATP hydrolysis-dependent translocation of MutS/MSHaway from the mismatch-containing site (10, 23).

As in S. cerevisiae, four MutL homologs have been identified in mammaliancells and are named PMS1, PMS2, MLH1, and MLH3. Unfortunately, the parallelnames used for the yeast and mammalian MutS homologs do not extend to all ofthe MutL homlogs, and this has been a source of confusion. As in yeast, the centralmammalian MutL homlog is named MLH1 and forms pairwise interactions withthe remaining three mammalian MutL homologs (138, 139, 179). The mammalianPMS2 is the homolog of yeast PMS1 and a heterodimer of MLH1 and PMS2(“MutLα”) is the major player in MMR. Mammalian PMS1 exhibits homology toyeast MLH2 and MLH3, and mammalian MLH1/PMS1 heterodimers are referredto as MutLβ (179). Finally, the recently identified mammalian MLH3 protein ismost similar to yeast MLH3, and was named on this basis (139). The roles ofthe MutLβ and the MLH1/MLH3 complexes in mammalian MMR have not been

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characterized, although overproduction of an MLH3 N-terminal deletion protein isassociated with a microsatellite instability phenotype (139). The observation thatMlh1-/- mice develop more tumors and exhibit slightly higher mutation rates thandoPms2-/- mice is consistent with a minor role for the MutLβ and MLH1/MLH3complexes in mammalian MMR (175, 239).

In addition to the mammalian MutS and MutL homologs, a human homolog ofyeast EXO1 has been identified and has been shown to interact with human MSH2(105, 192). A role of EXO1 in mammalian MMR has not yet been demonstrated,however. Finally, PCNA can be co-immunoprecipitated with MSH2, MLH1, andPMS2 from human cells (91), suggesting that PCNA also may play a role inmammalian MMR, perhaps being important in the strand discrimination process.

Invertebrates Genes encoding homologs of the yeast MSH and MLH proteinshave been identified in several multicellular invertebrate model systems, but thefunctions of the corresponding proteins have not been analyzed in detail. FourMutS homologs have been identified in the wormCaenorhabditis elegans: MSH2,MSH4, MSH5, and MSH6, so named based on homology to the correspondingyeast MSH proteins (LM Frisse & WK Thomas, personal communication; 229,241). The MSH4 protein, encoded by the wormhim-14gene, has been the most ex-tensively characterized and is required for all crossing-over during meiosis (241),presumably functioning as a complex with MSH5. Recently, a mutation in the geneencoding MSH2 has been identified (P Greenwell, J Culotti, & T Petes, personalcommunication). Homozygous mutantmsh-2strains have elevated microsatelliteinstability, but relatively normal fertility and chromosome disjunction. Interest-ingly, theC. elegansgenome does not appear to encode a homolog of either yeastMSH1 or MSH3 (LM Frisse & WK Thomas, personal communication). Theapparent lack of an MSH3 homolog inC. elegans(and Drosophila; see below)suggests that the MMR systems of worms and flies may be fundamentally dif-ferent from those of other eukaryotes. If this is indeed the case, then one mightexpect to see identical phenotypes resulting from loss of either MSH2 or MSH6in these organisms. In addition to the four MutS homlogs, there appear to be atleast three MutL homologs inC. elegansthat are related to yeast MLH1, MLH3,and PMS1 (LM Frisse & WK Thomas, personal communication; 139). Mutationsin the corresponding genes have not been identified.

In Drosophila melanogaster, the locusspellchecker1 (spel1)encodes an MSH2homolog, and as expected based on the yeast and mammalian data, flies with mu-tations inspel1exhibit an increased rate of microsatellite instability (81). Analysisof the Drosophila genome sequence has led to the identification of homologs of theyeast MSH6, MLH1, and PMS1 proteins (3, 82), and these proteins presumablyhave functions similar to those found in other eukaryotes. Surprisingly, Drosophilaappears to lack the meiotic-specific MSH4 and MSH5 proteins, and also does notappear to possess homologs of MSH1, MSH3, MLH2, or MLH3. In additionto the MutS and MutL homologs, the Drosophilatoscagene encodes an EXO1homolog, which seems to be female-specific in its effects (53). Finally, themei-9

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gene impacts meiotic PMS and crossing-over as well as DNA damage sensitivity,and, as noted above, the encoded protein is related to theS. cerevisiaenucleotideexcision repair protein RAD1 (194).

Plants Six genes encoding MutS homologs have been identified in theArabidopsis thalianagenome:AtMSH1, AtMSH2, AtMSH3, AtMSH4, AtMSH6,andAtMSH7(4, 40–42; JB Hays, personal communication). AtMSH7 shares ex-tensive amino acid similarity with AtMSH6, and phylogenetic analysis suggeststhat these two proteins diverged early in eukaryotic evolution (40). AtMSH2 canform heterodimers with AtMSH3 and AtMSH6 in vitro, and the resulting com-plexes have mismatch recognition specificities similar to those exhibited by thecorresponding yeast and mammalian complexes (40). In addition, AtMSH2 formsheterodimers with AtMSH7, and this complex (MutSγ ) has a mismatch recogni-tion spectrum distinct from that of the other two AtMSH2-containing complexes(40). To date, only plants have been found to contain the MSH7 protein, suggest-ing that the AtMSH2-AtMSH7 complex recognizes a DNA distortion that is eitherunique to plants or is normally handled by other MSH2-containing complexes inother organisms. Overexpression of two different predicted “dominant negative”AtMSH2 mutant proteins in plants results in microsatellite instability, indicatinga direct involvement of AtMSH2 in the repair of replication errors (JB Hays, per-sonal communication). In addition to the five MutS homologs, there appear to beat least three MutL homologs encoded by theA. thalianagenome, but none hasbeen characterized functionally (119, 139; JB Hays, personal communication).

MISMATCH REPAIR IN MUTATION AVOIDANCE

The MMR system plays a key role in the elimination of mutational intermediatesgenerated during DNA synthesis and thus helps to insure that DNA replicationis a high-fidelity process. The potentially devastating effects of elevated muta-tion rates are best illustrated by the predisposition to tumor development that isassociated with MMR defects in mammals (28). Although generally consideredto be detrimental, elevated mutation rates in microorganisms can be advanta-geous when environmental conditions demand rapid adaptation. The in vivo anti-mutator specificities of MMR proteins generally have been deduced by comparingmutation rates and spectra for selected genetic markers in wild-type strains versusstrains defective in a specific MMR component. Below, the roles of the MMRsystem in mutation avoidance in bacteria, yeast, and mammals are summarized.

Mutation Avoidance in Bacteria

The in vivo repair specificity of theE. coli MMR system has been determined byanalyzing spontaneous dominant forward mutations in the N-terminal portion ofthelacI gene (187–189). InmutH, mutL, ormutSstrains, thelacI forward mutationrate is elevated approximately 200-fold relative to the rate in a wild-type strain.

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In a wild-type strain, single-base frameshift mutations comprise only 5% of thetotal mutation events but account for approximately 25% of the events in MMR-defective strains, with almost all of the frameshift events in the MMR-defectivestrains occurring in a 5N mononucleotide run. Frameshift-specific reversion as-says also have demonstrated high-level instability of a dinucleotide poly(GT) run(137), as well as 8N mononucleotide runs (184) in MMR-defective strains. Notonly does the MMR system appear to be particularly important for repairing DNApolymerase slippage events in simple repeated sequences, but the system also ex-hibits an approximately 15-fold bias for the repair of transition versus transversionbase substitution intermediates (187). In general, the in vivo repair specificities oftheE. coli MMR system are consistent with those deduced from transformationexperiments employing heteroduplex DNA molecules with defined mismatches(54, 55, 127, 170).

The evolution of high mutation rates via elimination of the MMR system hasbeen observed when bacterial cells are maintained under adaptive conditions formany generations, with the enrichment for mutators being viewed as a consequenceof the selection process (201). For example, if the mutation rate at a defined locus iselevated 100-fold in MMR-defective cells, selection for mutations at that locus willresult in a 100-fold enrichment of MMR-defective cells among the selected mutantpopulation. In fact, with direct, successive selections for multiple mutations, theresulting population of cells is composed predominantly of MMR-defective muta-tors (146). In addition to accelerating adaptation, a mutator phenotype generateslow levels of sequence divergence that impose a recombination barrier and maycontribute to bacterial speciation processes (223). Although a mutator phenotypecan be advantageous under selective conditions, it does have a measurable negativeimpact on bacterial cells that are maintained nonselectively for many generations,with the mutational load eventually impairing overall fitness (87).

The evolution of MMR-defective mutators under controlled laboratory condi-tions may be relevant to the emergence of bacterial pathogens, which must rapidlyadapt to new hosts. For example, over 1% of pathogenicE. coli andSalmonellaentericaisolates have a strong mutator phenotype (134) and over 30% of thePseu-domonas aeruginosaisolates that chronically infect the lungs of cystic fibrosis pa-tients are mutators (165a). In addition, high levels of phase variation in pathogenicmeningococci are associated with MMR defects (27). The phase variation resultsfrom “on/off” frameshift mutations in a 7C mononucleotide run, which, as notedabove, are greatly elevated in the absence of a functioning MMR system. Finally,the bacterial observations likely are relevant to the multistep mutational processnecessary for cancer development in mammals, and may serve as a model for therelationship between MMR defects and accelerated tumor formation (see below).

Mutation Avoidance in Yeast

A variety of different forward and reverse mutation assays have been used toexamine the in vivo repair specificities of the MutS homologs inS. cerevisiae.

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Whereas reversion assays generally detect only a specific type of mutational event,forward mutation assays detect all events that impair gene function. Examples ofcommonly used reversion assays include thecyc1reversion system, which moni-tors all possible base substitutions within a single codon (98); thehom3-10rever-sion system, which detects mostly−1 events in a short mononucleotide run (150);and thelys21Bgl/lys21A746reversion systems, which detect a wide variety offrameshift mutations (90, 99). Forward mutations usually are monitored using theSUP4-oor CAN1gene (see 150, 238), with selection either for loss of tRNA sup-pressor activity or for resistance to canavanine, respectively. In addition, severalsystems have been developed to examine frameshift-generating DNA polymeraseslippage events that occur specifically in simple tandem repeats. Two widely usedsystems employ gene fusions to either the yeastURA3 or bacteriallacZ gene,with slippage events being identified by selecting for loss of URA3 function or byrestoration ofβ-galactosidase activity, respectively (see 204). Finally,LYS2-basedsystems have been developed that monitor the stabilities of mononucleotide runsof varying size and composition (101, 217). This latter type of system has beenparticularly useful for detecting subtle mutator phenotypes, as mutation rates inlong mononucleotide runs (>10N) are elevated 3 to 4 orders of magnitude whenthe yeast MMR system is completely inactivated (217).

Genetic data have demonstrated that MSH2 is required for all mismatchcorrection in nuclear DNA, while MSH3 and MSH6 are involved in the repairof distinct subsets of mutational intermediates. In thecyc1base substitution assay,msh2andmsh6mutants have identical mutator phenotypes, whilemsh3mutantsexhibit no detectable increase in mutation rates (63). Although these data indicatethat only the MSH2-MSH6 complex is involved in removing base substitution in-termediates, transformation experiments with mismatch-containing heteroduplexDNA suggest that the MSH2-MSH3 complex may have a minor role in repairingbase-base mismatches in some strains or sequence contexts (140). Consistent witha minor role in recognizing base-base mismatches, MSH3 is important for inhibit-ing recombination between diverged sequences that contain only base substitutionmutations (162). An important result that emerged from thecyc1studies was thevery large, MMR-associated difference in reversion rates of alleles that can re-vert via the same intermediates (63). For example, both GC to TA and TA to GCtransversions involve G/A or C/T mismatches as mutational intermediates, but theformer type of mutation occurs 1000-fold more frequently than the latter. It wassuggested that the reversion rate biases could be explained if the MMR system iscritically important for removing normal bases inserted opposite oxidatively dam-aged bases (e.g. 8-oxo-guanine or GO) in the DNA template. Subsequent work hasconfirmed this prediction by (a) demonstrating synergistic interactions between theMSH2or MSH6gene andOGG1, a gene whose product specifically removes GOlesions from DNA and (b) demonstrating high-affinity binding of MSH2-MSH6to GO/A mispairs (161).

In frameshift-specific assays,msh3or msh6mutants generally have only weakmutator phenotypes, while themsh3 msh6double mutant exhibits a very strong

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mutator phenotype equivalent to that of anmsh2mutant (90, 99, 122, 150, 199).Experiments in which the size of a simple repeat was varied from 1 bp to 20 bphave shown that the MSH2-MSH6 and MSH2-MSH3 complexes compete for therepair of frameshift intermediates containing extrahelical loops of 1 or 2 nt, butthat only the MSH2-MSH3 complex has repair activity against larger loops (199).Although this study indicated that extrahelical loops greater than approximately15 nt were not subject to MMR (see also 215), use of a different assay systemsuggests that a loop of approximately 100 bp can be removed by MSH2-MSH3(99). The minor discrepancies in mutation data obtained using different assayscan be resolved if one assumes that sequence context is an important componentof the mismatch recognition and/or repair process. Results obtained with in vitromismatch-binding assays (151) and in vivo homopolymer run assays (101) indeedindicate that sequence context can have a profound effect on MMR efficiency.

In both theCAN1 (150) andSUP4-o (238) forward mutation assays, theframeshift mutations show a large proportional increase relative to base substitu-tions when one compares themsh2spectra to spectra obtained in wild-type strains.This indicates that, as inE. coli, the yeast MMR system more efficiently correctsframeshift intermediates than it does base substitution intermediates. Further-more, experiments with frameshift-specific assays indicate that the yeast MMRsystem, like theE. coli system, more efficiently removes mutational intermedi-ates in reiterated sequence than in non-iterated sequence (90, 150). In contrast toE. coli, where the MMR system exhibits a strong preference for the repair oftransition over transversion base substitution intermediates, experiments with theSUP4-osystem suggest that the yeast MMR system exhibits a very weak bias forthe repair of transversion intermediates (238).

Both of the yeast MutS complexes function together with MutL components tocorrect mutational intermediates. In mutation rate assays, the phenotypes ofmlh1,pms1, andmsh2mutants generally are indistinguishable (90, 99, 101, 204), indicat-ing that the majority of mismatches repaired by MSH2-MSH3 and MSH2-MSH6complexes require a heterodimeric complex of the MutL proteins MLH1 andPMS1. Minor activities of the two additional MutL homologs (MLH2 and MLH3)have been identified, however, and both are assumed to work as heterodimericcomplexes with MLH1 to correct distortions recognized by the MSH2-MSH3complex (83, 102). Interestingly, only thePMS1mRNA is cell-cycle regulated inyeast (128, 202), raising the possibility that different MLH1-containing complexesmight exist at different times during the cell cycle. Thus the MLH1/PMS1 com-plex may predominate during DNA synthesis and deal with most replication errors,while the other complexes may be more important for repairing DNA distortionsthat arise out of the context of normal DNA replication.

Most mutation studies in yeast have focused on the genetic consequences ofcompletely eliminating a particular MMR protein. Experiments indicate, how-ever, that the correct stoichiometry between MMR components also is important.MSH2/msh2andMLH1/mlh1heterozygous diploids, for example, exhibit a weakmutator phenotype (58, 195), and overproduction of wild-type MLH1 can result

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in a mutator phenotype that is very similar to that of anmlh1null mutant (195). Inthe case ofMLH1/mlh1heterozygotes, the mutator phenotype results from loss ofthe wild-type gene in a small proportion of cells, and this loss can be stimulated byDNA damaging agents (195). In addition to stoichiometry effects, some non-nullmutant proteins can impair the MMR system in the presence of the correspondingwild-type protein (i.e. dominant negative effects). Dominant negative mutations inMSH6(25, 47) andMSH2(9, 58, 205, 206) have been identified, and the resultingproteins are presumed to form nonproductive complexes that either preclude theformation of repair-competent complexes or bind nonproductively to mismatchesand thereby block potential repair.

Mutation Avoidance in Mammals

The knowledge of simple repeat instability in MMR-defectiveE. coli and yeastwas critical for linking the characteristic instability of simple repeats (microsatel-lite instability or MSI phenotype) in some types of human cancer to MMR defects.Most notably, mutations inMLH1 andMSH2are associated with 60% to 70% ofthe cases of hereditary non-polyposis colon cancer (HNPCC); a very small numberof HNPCC cases also are associated with mutations inPMS1, PMS2, andMSH6[reviewed in (28)]. Although there have been no reported cases of HNPCC result-ing from mutations inMSH3or MLH3, the mouseMLH3 gene physically mapsto the colon cancer susceptibility I locus (139). Individuals heterozygous for arelevant MMR gene mutation are predisposed to HNPCC and, in a manner anal-ogous to tumor suppressor genes, loss of heterozygosity (or loss of expression ofthe functional gene) leads to highly elevated mutation rates and subsequent tumordevelopment (104). In addition to HNPCC, defects in MMR have been associatedwith sporadic colorectal, endometrial, and gastric carcinomas. Unlike the HNPCCcases, however, the majority of these sporadic carcinomas do not have identifiablemutations in eitherMLH1 or MSH2, but rather epigenetic transcriptional silenc-ing has been linked to the MSI phenotype. Specifically, hypermethylation of theMLH1 promoter has been associated with a complete loss of protein expression(43, 68, 79, 107, 135, 200, 220). In addition to functional inactivation of the mam-malian MMR system by genetic or epigenetic mechanisms of protein loss, MMRdefects can result from overexpression of the MSH3 protein via co-amplificationof the MSH3gene with the adjacentDHFR locus (59, 149). The excess MSH3completely sequesters MSH2, thus preventing the formation of MSH2-MSH6complexes and producing the equivalent of anMSH6mutant. Finally, an HNPCC-associatedPMS2nonsense mutation has been identified that exerts a dominantnegative effect (164).

Spontaneous mutation rates and spectra for theHPRTgene have been obtainedfrom MMR-defective human tumor cells lines (144, 165, 219). Forward mutationsin theHPRTgene are elevated several hundred-fold in human tumor cell lines thatare deficient in MLH1 or MSH2, and one sees a proportional increase in+1 and−1frameshift mutations relative to base substitutions. As in MMR-defective yeast,

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mononucleotide runs are hotspots for frameshift events in tumor cells, indicatingthat these sequences are particularly “at risk” for accumulating protein-inactivatingmutations. Indeed, frameshifts in mononucleotide runs within the APC and TGF-β

genes, both of which are important in colon tumorigenesis, have been identified intumor cells with the MSI phenotype (111, 148). The human tumor cell line HHUAis deficient in both MSH3 and MSH6, and chromosome transfer experiments haveallowed the generation of cell lines deficient in only one of the proteins (219).Examination of microsatellite instability andHPRTmutations in these cell linesindicates that MSH6 can repair extrahelical loops that are 1–4 nt in size whileMSH3 removes loops that are 2–4 nt in size. This contrasts with the yeast MMRsystem, where MSH3/MSH6 functional redundancy is seen for 1–2 nt loops, butonly MSH3 is capable of repairing loops larger than 2 nt (see above). Surprisingly,the HHUA derivative that is presumed to be only MSH3-defective has an elevatedrate of base substitution mutations. This suggests that, in human cells, MSH3 mayparticipate in the correction of base substitution intermediates.

The mammalian MMR system has been implicated in somatic hypermutationof the immunoglobulin variable genes, a process that generates high-affinity anti-bodies in response to antigen stimulation. Some studies have indicated that fulllevels of hypermutation require a functioning MMR system (29, 178), whereasother studies have demonstrated that the spectrum, but not the frequency, of hy-permutation is altered in MMR-deficient mice (173, 230). Finally, some studieshave reported no involvement of the MMR system in the process of somatic hy-permutation (84, 118). Recent data have demonstrated a severe perturbation of theB cell response to antigen stimulation in MMR-deficient mice, and it has beensuggested that the associated sampling biases could account for the conflictingresults obtained when assessing somatic hypermutation (221).

As models for human disease, mouse knockouts ofMSH2, MSH3, MSH6,PMS1, PMS2, or MLH1 have been constructed and all, with the exception ofPMS1- and MSH3-deficient animals, show an increased occurrence of diversetypes of internal organ tumors (14, 50, 51, 66, 175). As predicted by genetic stud-ies in yeast,Msh6-/- mice are not completely MMR-defective and have a differentspectrum of tumors thanMsh2-/- mice, while the double knockoutMsh6-/- Msh3-/-mice are identical toMsh2-/-mice (51). Interestingly,Mlh1-/- andPms2-/- micehave different spectra of tumors (175) and exhibit subtle differences in mutationrates (239), both of which presumably reflect minor roles of MLH1-PMS1 andMLH1-MLH3 in mutation avoidance. Mutation rates and spectra ofMsh2-/- orPms2-/- knockout mice containing either alacI or SUPF forward mutation re-porter system, respectively, have been reported (11, 19, 159). In contrast to thetissue-specific tumor development observed inPms2-/- mice, all tissues that wereexamined exhibited comparable increases inSUPF mutation frequency (159).The prominence of frameshift mutations in the spectrum from thePms2-/- miceis consistent with the general consensus that the MMR machinery plays the pri-mary role in the correction of DNA polymerase slippage errors. ThelacI mutationassay detects primarily base substitutions, and, as expected, mutation frequencieswere elevated inMsh2-/- mice(11). Interestingly, a comparison of tumor tissue and

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normal tissue from these mice revealed a higher mutation frequency in the tumortissue, although the mutation spectra were indistinguishable (19). This suggeststhat additional destabilizing mechanisms may be important in tumor developmentin MMR-defective mice, but the relevance of this to human cancer is unclear.

Given the inherent limitations of mammalian systems, a number of studieshave used yeast to model the genetic effects of human mutations associated withHNPCC. First, amino acid changes analogous toMSH2, MLH1, andPMS2alter-ations in HNPCC kindreds have been introduced by site-directed mutagenesis intothe homologous yeast proteins (36, 167, 195). The resulting loss of MMR activityin haploid yeast strains has confirmed a causative role of the human mutations inHNPCC. In addition to introducing HNPCC-associated changes into yeast pro-teins, isolation of dominant negative alleles of yeastMSH2has uncovered aminoacid alterations analogous to those in HNPCC patients (205). A second type of ex-periment that has been used for modeling HNPCC in yeast is to assess the potentialimpact of heterozygous HNPCC mutations in diploid yeast cells. As noted previ-ously, a highly sensitive reporter assay has been used to demonstrate a weak mutatorphenotype ofMLH1/mlh1andMSH2/msh2diploid cells relative to homozygouswild-type strains (58, 195). This assay has also been used to detect dominant neg-ative effects of HNPCC alleles. A third general approach for modeling HNPCCmutations in yeast is based on the ability of wild-type human MMR proteins tointerfere with the function of the endogenous yeast proteins and thereby produce astrong mutator phenotype (36, 197). Loss of the dominant negative phenotype as-sociated with wild-type human MLH1, for example, has been exploited as a methodto rapidly and inexpensively identify missense mutations in candidateMLH1 HN-PCC alleles (197). In the case of the human MutS homologs, co-expression ofhuman MSH2 along with either human MSH6 or MSH3 generates a dominantnegative phenotype in yeast, indicating that the MutSα and MutSβ complexesinterfere with the function of the yeast MMR machinery, presumably by nonpro-ductive mismatch binding (36). An HNPCC-associatedMSH2mutation elimi-nates the dominant-negative effect, thus demonstrating the potential utility of theco-expression system for characterization of suspected disease-associated alleles.

ROLES OF MMR IN RECOMBINATION PROCESSES

Homologous recombination involves the pairing of complementary single strandsderived from two different duplexes, resulting in a heteroduplex DNA molecule.If the recombining duplexes are not identical in sequence, then the resultingheteroduplex will contain mismatches, and these mismatches can be recognizedand processed by the same MMR machinery that removes DNA replication errors.Interaction of MMR proteins with mismatch-containing recombination intermedi-ates can trigger one of two events: simple mismatch correction or complete abortionof the recombination event. In addition, MMR proteins are involved in the removalof the nonhomologous single-strand tails that are generated during recombinationprocesses. These three recombination-related activities are discussed below.

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Mismatch Correction in Recombination Intermediates

In prokaryotic organisms, mismatch-containing heteroduplex intermediates areformed during the processes of transduction, transformation, and conjugation.Nick-directed repair of such mismatches by the MMR machinery has been invokedto explain marker- or allele-specific variations in recombination frequencies [re-viewed in (37)]. InStreptococcus pneumoniae, for example, the differences in thetransformation efficiencies of high-efficiency versus low-efficiency donor mark-ers disappears in MMR-deficient strains. The MMR system presumably degradessome or all of the donor DNA strand when well-repaired mismatches are presentin the heteroduplex recombination intermediate, thus resulting in low transforma-tion efficiencies. If mismatches are not repaired, segregation of the mismatchedstrands at the next round of DNA replication will give rise to a recombinant. Asimilar phenomenon may occur during yeast transformation, where eliminationof the MMR machinery increases the frequency with which mutant markers areintroduced into the genome (136, 186).

In eukaryotes, the repair of mismatches in recombination intermediatesresults in the genetic phenomenon of gene conversion, in which information onone chromosome is replaced with information from the homologous chromosome[reviewed in (171)]. In organisms such as yeast, where it is possible to analyzeall four products derived from a single meiosis, a non-Mendelian 3:1 segrega-tion pattern of allelic sequences is diagnostic of a gene conversion event. In theabsence of a functional MMR system, mismatches in heteroduplex recombina-tion intermediates are not repaired, and the mismatch-containing strands segre-grate at the first mitotic division following meiosis (postmeiotic segregation orPMS). In yeastmsh2, pms1, ormlh1mutants, which are essentially defective in allmismatch repair, meiotic gene conversion events are reduced while PMS eventsare elevated (8, 176, 181, 227). The repair patch presumably is small relative tothe extent of meiotic heteroduplex DNA formed, so the repair process does notnecessarily interfere with the formation of mature recombinants. Some meiotic dataindicate, however, that mismatches may impact the progression of recombinationin an MMR-dependent manner (8, 52), and this regulatory activity may dependon when mismatches are detected during the recombination process. Althoughthe same basic MMR machinery detects mismatches in both recombination andreplication intermediates, there likely are accessory proteins unique to each typeof repair. For example, PCNA appears to play a role in correcting DNA repli-cation errors in yeast (120, 218) but has not been implicated in the correction ofheteroduplex recombination intermediates.

Regulation of Recombination Between NonidenticalSequences (Anti-Recombination)

DNA sequence divergence interferes with homologous recombination in bothMMR-dependent and MMR-independent manners (48, 222), and the focus here is

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on the MMR-dependent inhibition of recombination. The potent anti-recombinationactivity of MMR proteins was first demonstrated in conjugational crosses betweenE. coliandSalmonella typhimurium, the genomes of which exhibit approximately15% sequence divergence. Elimination of the recipient MMR system is associ-ated with up to a 1000-fold increase in conjugational recombination frequencies,with the effect being much stronger inmutSandmutL mutants than inmutH oruvrDmutants (180). The anti-recombination activity of the MMR machinery is notlimited to conjugational crosses but also impacts plasmid-λ recombination (196)and intrachromosomal deletion formation (172) inE. coli; intrachromosomal re-combination inS. typhimurium(1); transduction between different Salmonellaspecies (240); and transformation inS. pneumoniae(143). Although the exceptionto this generality seems to be transformation in Bacillus, transformation in this or-ganism normally takes place under starvation conditions, and thus at a time whenthe MMR machinery normally may be inactivated (142).

Two general models have been proposed to explain the anti-recombinationactivity of the bacterial MMR system (see 180). The heteroduplex destructionmodel involves mismatch-stimulated killing of a recombination intermediate dueto the accumulation of multiple MMR-generated nicks. The heteroduplex rejectionmodel is nondestructive in that there is no cleavage of recombination intermediates,and proposes instead that mismatch recognition leads to a reversal of the recombi-nation process. The transformation efficiency intoE. coli of plasmids containinga heteroduplex region with 18% mismatches is not greatly impacted by the MMRmachinery, suggesting that the MMR system operates primarily by impeding orreversing the formation of recombination intermediates rather than by destroyingpreformed heteroduplex intermediates (226). Also, in vitro studies demonstratingthe blockage of RecA-mediated strand exchange by MutS and MutL are consistentwith the anti-recombination role the MMR system being exerted during the for-mation of heteroduplex DNA (231, 232). Finally, recent genetic data indicate thatthe MMR machinery inE. coli edits the fidelity of recombination at two steps: anearly step that is MutH-independent and a late step that is MutH-dependent (203).The early editing step requires MutS, MutL, and UvrD, and is hypothesized toinvolve mismatch-triggered, helicase-mediated reversal of heteroduplex forma-tion. The ejected single strand may then be a substrate for the RecBCD nuclease,which would prevent reiterative attempts to complete the recombination process(240). The late editing step is dependent on MutH and requires de novo DNAsynthesis, and presumably operates on mismatches linked to unmethylated GATCsequences via replicative extension of the invading 3′ end.

The anti-recombination activity of the eukaryotic MMR machinery has beenbest characterized inS. cerevisiae, where it can inhibit recombination betweendiverged sequences both in mitosis and in meiosis [reviewed in (100)]. Using modelrecombination substrates, it has been demonstrated that a single mismatch is suffi-cient to inhibit recombination in an MMR-dependent manner, with additional mis-matches having a cumulative negative effect on recombination rates (33, 48). Ata mismatch density of several percent, however, the probability of a heteroduplex

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recombination intermediate escaping detection by the MMR system becomes es-sentially zero, so that additional mismatches fail to elicit more anti-recombinationactivity. Analyses of the products of recombination between diverged sequencesgenerally have supported a role for the MMR machinery in limiting (either byblockage or reversal) heteroduplex formation in a mismatch-dependent manner(32, 33, 160, 208), although one study suggests that a high mismatch density actu-ally may increase the length of heteroduplex DNA (163). Finally, sequence diver-gence inhibits not only standard homologous recombination, but also reduces theefficiency of the single strand annealing recombination pathway [(207); see belowfor a description of this pathway].

The genetic requirements of anti-recombination in yeast have been examinedusing substrates containing defined types of mismatches, and, in general, theMSH2-MSH3 and MSH2-MSH6 complexes recognize the same types of mis-matches in recombination intermediates as they do in replication intermediates(162). Althoughmsh2andmlh1or pms1mutants have virtually identical pheno-types in mutation assays,msh2mutants consistently exhibit higher recombinationrates between diverged sequences than dopms1or mlh1mutants (33, 162). Thisindicates that mismatch binding alone is sufficient to elicit some inhibition ofrecombination in yeast, which is consistent with in vitro results demonstratingthe blockage of RecA-mediated strand exchange by the bacterial MutS protein(231, 232). The nonequivalent roles of the yeast MSH and MLH proteins in anti-recombination are even more striking during single strand annealing, with MLHproteins playing at most a minor role in the mismatch-related suppression of thistype of recombination (N Sugawara, B Studamire, E Alani & JE Haber, personalcommunication). In contrast to the lack of involvement of the RAD1-RAD10 nu-cleotide excision repair complex in MMR-associated mutation avoidance, theseproteins have a clear role in regulating recombination between diverged sequences(162). Whether the anti-recombination role of RAD1-RAD10 is structural or en-zymatic remains to be determined. Finally, EXO1 plays a minor role in inhibitingrecombination between diverged sequences (162).

The anti-recombination activity of prokaryotic MMR systems is thought toconstitute an effective barrier to DNA transfer between different species and tobe important in establishing a genetic barrier during speciation processes (222).An examination ofE. coli lines evolved independently in the laboratory from acommon ancestor indeed indicates that loss of the MMR system accelerates se-quence divergence via the enhanced rate of mutation accumulation. This sequencedivergence then becomes a barrier to recombination with the ancestral line uponreintroduction (via recombination) of a functioning MMR system (223). In yeast,the MMR system inhibits crossing-over between diverged chromosomes in in-terspecific hybrids during meiosis (30, 113), and hence also may be importantfor establishing or maintaining genetic barriers between species. In addition, themismatch-triggered anti-recombination activity of eukaryotic MMR proteins likelyinhibits ectopic interactions between diverged, repetitive DNA sequences duringboth mitosis and meiosis, and thereby promotes genome stability by preventingdeleterious genome rearrangements. Interestingly, mitotic recombination in yeast

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is more sensitive (in an MMR-dependent manner) to low levels of sequence di-vergence than is meiotic recombination (33), which may help reinforce the strongbias for sister chromatid versus interhomolog interactions during mitosis. Theobservations made in yeast are likely relevant to the mismatch-associated regula-tion of recombination in higher eukaryotes, as gene targeting studies in mice havedemonstrated anti-recombination roles for MSH2 and MSH6 (50, 51).

In considering the regulatory roles of MMR proteins in eukaryotic recombina-tion processes, a final issue that deserves mention is the relationship between themismatch correction role of MMR proteins, which is responsible for gene con-version events, and the anti-recombination role of MMR proteins, which activelydissuades interactions between nonidentical sequences. One attractive idea is thatanti-recombination activity is exerted during the very early stages of recombina-tion, perhaps during the initial homology search or strand exchange reaction. Thisediting function could involve direct interactions between MMR proteins and com-ponents of the recombination machinery. Consistent with such a scenario, recentdata have demonstrated an association of yeast MSH2 with molecules that have sus-tained an HO-induced double strand break in vivo (69). Whether a mismatch(es)triggers anti-recombination is a probability issue (see 48), so that low levels ofdivergence would reduce, but not prevent the formation of stable recombinationintermediates. Once formed, mismatches in a stable recombination intermediatewould again be subject to detection by the MMR machinery, but in this case, theoutcome would be a simple repair process. This latter detection could be linked tothe replicative extension of the invading 3′ end, or could occur at an even later step.Finally, the in vitro binding of MMR proteins to Holliday junctions (7, 152) raisesthe possibility that MMR proteins may stabilize these structures, which in turncould promote the process of crossing-over. One study suggests that the presenceof the yeast MMR machinery indeed increases the association of crossovers withHO-induced gene conversion events (116).

Nonhomologous Tail Removal

Studies inS. cerevisiaehave identified roles of MSH2 and MSH3 in bothhomologous recombination and in a specialized type of recombination knownas single strand annealing [reviewed in (168)]. In homologous recombination, asingle-stranded tail with a 3′ end invades a homologous duplex DNA molecule,and the invading 3′ end is then used to prime DNA synthesis. If the 3′ end itselfis not homologous to the invaded duplex, the nonhomologous segment must beremoved before DNA synthesis can be initiated. In the single strand annealing(SSA) recombination pathway, a double-strand break between directly repeatedsequences is acted on by a 5′ to 3′ exonuclease to yield long tails with 3′ ends.Exposure of complementary sequences derived from the flanking direct repeats isfollowed by strand annealing, removal of the unpaired 3′ tails, and ligation of re-maining nicks. In both homologous recombination and SSA, the unpaired 3′ tailsare removed by the RAD1-RAD10 endonuclease (74), which is best known forits role in the nucleotide excision repair pathway where it nicks a damaged DNA

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strand 5′ of the damage (18). Although yeast MMR proteins play no apparent rolein nucleotide excision repair, genetic studies have demonstrated that MSH2 andMSH3 cooperate with the RAD1-RAD10 endonuclease to effect removal of thenonhomologous 3′ tails generated during mitotic homologous recombination andSSA (186, 207). This “clippase” role appears to be unique to the MSH2-MSH3complex, as 3′ tail removal is not impaired inmsh6, pms1or mlh1mutants (207).Interestingly, mutant MSH2 proteins have been identified that uncouple the role ofthe protein in replication error correction from its clippase role (205), and this un-coupling presumably reflects interactions with different downstream componentsin the recombination versus MMR pathways.

In keeping with the binding of the MSH2-MSH3 complex to distorted DNAstructures, it has been proposed that MSH2-MSH3 binds to and stabilizes branchedstructures with 3′ tails, thus facilitating cleavage by the RAD1-RAD10 endonu-clease at the junction of single standed and duplex DNA (207). Two-hybrid andco-immunoprecipitation studies have demonstrated that MSH2 indeed interactswith RAD1 and RAD10, but it also interacts with a number of additional nu-cleotide excision repair proteins (22). The relevance of these latter interactions isnot clear, as RAD1 and RAD10 are the only nucleotide excision repair proteinsthat play a role in nonhomologous tail removal (117). In addition to nonhomolo-gous 3′ tail removal, genetic studies indicate that RAD1 and MSH2 also cooperateto remove large loops in heteroduplex meiotic recombination intermediates (126)and mutational intermediates (99), and this activity may be related to the mitoticMSH2-MSH3, RAD1-RAD10 clippase activity. Finally, studies withMsh2-/- micesuggest that MSH2 plays a role in promoting immunoglobulin class switching inmammals (67, 193). Although this also may reflect a clippase function similar tothat observed in yeast, class switching is reduced not only inMsh2-/- cells, butalso in cells fromMlh1-/- andPms2-/- mice (193).

ROLES OF MMR PROTEINS IN DNA DAMAGE-RELATEDPROCESSES

Prokaryotic and eukaryotic MMR proteins recognize numerous types of DNAdamage and either initiate or directly participate in the ensuing cellular responsesto the damage. The most widely studied damage-related phenomenon is that ofalkylation tolerance, which was initially described for MMR-defective bacterialcells (123) and subsequently was found to be characteristic of MMR-defectivemammalian cells (26, 124). Alkylation damage usually is induced using N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) or N-methyl-N-nitrosourea (MNU), bothof which generate O6-methyl-guanine (O6-MeG) as the most relevant lesion. Inkeeping with the known activities of MMR proteins, insertion of a base oppositeO6-MeG is suggested to initiate a futile cycle of mismatch repair (i.e. repetitiveexcision and resynthesis of the newly synthesized DNA strand using the dam-aged strand as a template), which ultimately leads to cell death. In the absenceof a functional MMR system, O6-MeG is tolerated by the cell and becomes a

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miscoding/mutagenic lesion rather than a lethal lesion. Curiously, MMR-defectiveyeast cells do not exhibit increased tolerance to alkylating agents (20), althoughinactivation ofMLH1, MLH2, MSH2, MSH3, or MSH6(but notPMS1) has beenreported to increase resistance to some anticancer drugs (62). Furthermore, flieslacking thespel1gene do not exhibit increased tolerance to methylating agents(81).

In mammalian cells, MNNG triggers an MMR-dependent G2-M arrest (103),which is followed by induction of an MMR-dependent apoptotic response (214). Inaddition to being necessary for O6-MeG-triggered apoptosis, mammalian MMRproteins are capable of binding a wide array of lesions in vitro (61, 153, 233),and presumably are important for triggering apoptosis in response to a varietyof DNA damaging agents (see 80, 130). Although only MutSα and MutLα areinvolved in the alkylation damage response (51, 60, 108), MutSβ may be impor-tant for triggering apoptosis in response to other types of DNA damage (233).Recent data indicate that the MMR-associated apoptotic response has both p53-dependent and p53-independent components (45, 60, 108, 233), but the precisemechanism whereby the MMR system induces apoptosis is not understood. Onepossibility is that MMR components act as general damage sensors and directlytransduce the damage signal to downstream signaling components. The recentdescription of a BRCA1-associated genome surveillance complex (BASC) thatcontains MSH2, MSH6, MLH1, and other DNA repair/metabolism proteins wouldbe consistent with a damage sensor role (225). Alternatively, the role of the MMRsystem may be in damage processing, in which case the resulting nicks or gapswould be the signals that trigger apoptosis. Regardless of the mechanism ofMMR-associated apoptosis, this phenomenon has profound implications for themanagement of cancer, where a standard approach is to use DNA-damaging agentsto selectively induce apoptosis in tumor cells. Both radiation and chemotherapeuticagents induce lesions that may require a functional MMR system in order to be di-rected toward an apoptotic pathway, making MMR-defective tumor cells refractoryto this treatment. In addition, treatment of MMR-competent tumor cells with DNA-damaging agents may select for MMR-defective tumor cells (35), which not onlywill present an impediment to future attempts to selectively kill tumor cells, butalso will elevate mutation rates, possibly resulting in more aggressive tumor cells.

In addition to promoting the toxicity associated with alkylation damage, bac-terial and mammalian MMR proteins, but not yeast MMR proteins, are requiredfor the transcription-coupled repair (TCR) of UV damage that occurs via thenucleotide excision repair pathway (154, 155, 209). Transcription-coupled repairrefers to the preferential removal of lesions from the transcribed strands of activegenes, and this process is triggered by damage-induced blockage of RNA poly-merase progression [reviewed in (185)]. How MMR proteins facilitate TCR isnot known, but a likely step would relate to DNA damage recognition. Althoughyeast MMR proteins are not required for TCR of UV damage (209), disruption ofthe yeast MMR pathway has been reported to increase the UV sensitivity of cellsthat are completely defective in the nucleotide excision repair pathway (22, 177).This suggests the existence of a minor MSH2-dependent pathway for the repair of

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UV-induced damage in yeast. Finally, TCR has been extended to the removal ofoxidative lesions, and some MMR proteins are important in this process in bothmammalian and yeast cells (132, 133).

High doses of mutagenic agents that typically induce only base substitutionmutations have been reported to induce frameshift mutations inE. coli, withthe types of frameshifts observed being those typical of MMR-defective cells(e.g. frameshifts in mononucleotide runs). One interpretation of these data is thathigh levels of DNA damage can saturate the bacterial MMR system, and therebyprevent the participation of MMR proteins in the removal of replication errors(44). A similar saturation of eukaryotic MMR systems by DNA damaging agentshas not been reported but may be relevant to the potentially mutagenic effects ofDNA damaging agents used to combat tumor cells.

MEIOTIC-SPECIFIC ROLES OF MMR PROTEINS

Eukaryotic homologs of the bacterial MutS and MutL proteins have divergedfunctionally and some have acquired novel, meiotic-specific roles that are unrelatedto mismatch binding/repair. The first two such proteins identified were the yeastMutS homologs MSH4 and MSH5, each of which appears to have completelylost the ability to participate in a standard mismatch repair reaction. Instead, lossof either MSH4 or MSH5 is associated with an approximately 50% reduction inmeiotic crossing-over, resulting in increased levels of homolog nondisjunction anddecreased viability of meiotic products (109, 182). TheMSH4andMSH5genesare in the same epistasis group (109) and, like the other MutS homologs, physicallyinteract to form heterodimers (174). As gene conversion levels are normal inmsh4or msh5mutants, the MSH4-MSH5 complex is assumed to impact a late step inrecombination, perhaps promoting the formation or stability of Holliday junctions,which then can be processed into crossover events (for a discussion see 158).Based on the abilities of the MSH2-MSH6 and MSH2-MSH3 complexes to binddistorted DNA structures, it has been suggested that the MSH4-MSH5 complexmight exert its activity by directly binding Holliday junctions. Consistent witha role in processing recombination intermediates, MSH4 localizes in a punctatepattern to fully synapsed meiotic chromosomes (182). Although the yeast MSH4-MSH5 complex is generally assumed to function only in meiosis, a non-null alleleof MSH5has been identified that results in alkylation tolerance (20).

Just as the yeast MSH2-MSH3 and MSH2-MSH6 complexes interact with anMLH1-containing complex to complete the MMR reaction, genetic data indicatethat the MSH4-MSH5 heterodimer interacts specifically with the MLH1-MLH3complex to promote meiotic crossing-over. Bothmlh1andmlh3mutants exhibita reduction in meiotic crossover events (112, 224), and epistasis analysis indicatesthat MLH1 andMSH4act in the same crossover pathway (112). In addition tothe meiotic-specific role of MLH1-MLH3 complex,mlh2mutants exhibit a subtlemeiotic phenotype when non-Mendelian segregation patterns are analyzed (224).This latter observation suggests that the MLH1-MLH2 complex may play a meiotic

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role that is distinct from the heteroduplex repair role associated with the MLH1-PMS1 complex.

Homologs of yeast MSH4 and MSH5 have been identified inC. elegans(229, 241), mouse (65, 106), and humans (24, 105, 169, 229). In mammalian cells,expression of the corresponding genes is highest in the testis and ovaries(24, 105, 106, 169), which is consistent with a meiotic-specific function. Disrup-tion of theMsh5gene in the mouse does not impact viability, but homozygousMsh5-/- animals are sterile (65). In contrast to normal chromosome pairing andcompletion of meiosis inmsh4/msh4or msh5/msh5yeast cells (109, 182), meiosisis arrested in prophase I inMsh5-/- mice, with an accompanying loss of testicularand ovarian tissue (65). AlthoughC. elegansresembles yeast in that chromosomepairing appears normal in the absence of MSH4 (encoded by thehim-14gene),MSH4 is absolutely required for the production of meiotic crossover chromosomesin C. elegans(241).

As noted above, the yeast MLH1/MLH3 complex likely interacts with theMSH4-MSH5 complex to promote meiotic crossing-over, with the other yeastMutL homologs having functions related only to mismatch repair activity (224).In the mouse, however, the MutL homologs have a much greater impact on mei-otic processes. Both male and femaleMlh1-/- mice are sterile, and examinationof testicular tissue indicates that spermatocytes arrest in meiosis I with prema-turely separated chromosomes (15, 64). MLH1 localizes to the presumptive sitesof meiotic crossing-over in wild type mice, which is consistent with a role inrecombination (15). Interestingly,Pms2-/- male mice are sterile and exhibit ab-normalities in meiotic chromosome pairing, whereasPms2-/- female mice arefertile (14). The sex-specific meiotic defect in mice contrasts with the essen-tially normal meiosis that occurs in the correspondingpms1/pms1yeast mutants.Although there currently is no information on the meiotic roles of MutL homologsin organisms other than yeast and mice, studies in additional organisms shouldhelp clarify the meiotic roles of the eukaryotic MLH proteins.

SUMMARY AND FUTURE PERSPECTIVES

The model MMR system ofE. coli is important for repairing DNA replicationerrors, for mounting DNA damage responses, and for inhibiting recombinationbetween diverged sequences. Since the discovery of a link between MMR defectsand human cancer in 1993, there has been an explosion of MMR-related researchin eukaryotes. In eukaryotes, the single bacterial MutS and MutL proteins havebeen replaced with multiple homologs that form heterodimeric complexes thatnot only perform discrete functions in DNA repair processes, but that also haveevolved novel roles in meiotic chromosome metabolism. With the exception of themitochondrial yeast MSH1 protein, studies in yeast and mammals uncovered thesame basic repertoire of MSH and MLH proteins (MSH2-MSH6, MLH1-MLH3,and PMS1), suggesting that the general organization of the MMR system likelywould be conserved from lower to higher eukaryotes. Surprisingly, however, recent

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genome sequencing projects have failed to detect a MSH3 homolog in Drosophilaor C. elegans, or MSH4-MSH5 homologs in Drosophila. In addition, theA.thaliana genome has been found to contain a seventh MutS homolog (MSH7),which also forms a mismatch-binding complex with MSH2. More studies clearlyare needed to determine the function of novel MMR proteins such as MSH7, and itis possible that other novel MutS or MutL homologs may surface when additionalgenomes are sequenced. In addition, the absence of key MMR proteins fromC. elegansand Drosophila raises the issue of whether the respective roles of theseproteins are fulfilled by other MMR or non-MMR proteins.

MMR studies in eukaryotes have focused not only on the identification of MutSand MutL homologs, but, more importantly, on the functions of these proteins.Genetic studies, particularly those done in yeast, have revealed the roles of indi-vidual MMR proteins and complexes in promoting genome stability, particularlyin relation to mutation and recombination processes. These genetic observationsare of direct relevance to the genome destabilizing forces that underlie the devel-opment of cancer. Much of the biochemical research to date has focused on theinteractions between and functions of the individual eukaryotic MutS and MutLhomologs. While interactions between these proteins are clearly of functionalimportance, the MSH and MLH proteins do not exist in a vacuum, and likelyinteract with a large number of other DNA repair and metabolic proteins, someof which have been identified. Such interactions are assumed to be important inDNA damage responses and in the regulation of recombination, and also mayultimately provide the key to how strand discrimination is achieved during therepair of DNA replication errors. The complementary genetic and biochemicalapproaches that have been employed to elucidate the diverse functions of MMRproteins have been tremendously successful, and we can look forward to continuedrapid developments in our understanding of MMR processes.

ACKNOWLEDGMENTS

We thank Abhijit Datta, Eric Alani, and Rachelle Spell for critical commentson this review, and the many colleagues who communicated unpublished results.Support was provided by NIH and NSF grants to SJ-R.

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Figure 1 The bacterial paradigm for mismatch repair of DNA replication errors.

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DNA Mismatch Repair and Genetic Instability

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