The Components of Prokaryotic Transcription

MINI REVIEW

Structure and function of the components of the human DNA mismatch

repair system

Thomas Jascur* and C. Richard Boland

Department of Internal Medicine, Baylor Research Institute, Baylor University Medical Center, Dallas, TX

DNA mismatch repair (MMR) is one of the several enzyme sys- tems involved in DNA homeostasis. DNA MMR is involved in the repair of specific types of errors that occur during new DNA syn- thesis; loss of this system leads to an accelerated accumulation of potential mutations, and predisposes to certain types of cancers. Germline mutations in some of the DNA MMR genes cause the he- reditary cancer predisposition, Lynch syndrome. This review addresses advances in the biochemistry of DNA MMR and its rela- tionship to carcinogenesis. ' 2006 Wiley-Liss, Inc.

Key words: DNA mismatch repair; microsatellite instability; lynch syndrome; HNPCC; colorectal cancer; hMSH2; hMLH1; hMSH6; hPMS2; exonuclease I

The DNA mismatch repair (MMR) system was at one time in the exclusive domain of the microbial biochemist, but has been thrust into the mainstream of the cancer biologist because of its importance in carcinogenesis. Approximately 15% of colorectal cancers develop through mechanisms whereby this form of DNA maintenance is lost, leading to 100- to 1,000-fold increases in error rates during replication. This review of recent progress in DNA MMR research is intended for the tumor biologist who is interested in structural and functional details of this system. There have been several reviews of the clinical aspects of DNA MMR that emphasize the diagnostic uses of testing for microsatellite instability and the use of immunohistochemistry of the DNA MMR proteins in colorectal cancer.

1–7 This review will serve to

complement those reviews, and will highlight some of the bio- chemical issues that underlie DNA MMR.

Components of the DNA MMR system

The DNA MMR system corrects DNA base pairing errors in newly replicated DNA. The primary DNA polymerase in eukaryo- tic S phase DNA replication, polymerase d, has 30 > 50 proofread- ing activity that corrects 99% of replication errors. Nevertheless, mispaired nucleotides are occasionally left behind, as are small insertion/deletion mutations that are prone to occur at repetitive sequences. Microsatellites are multiple tandem repeats in which the repetitive element consists of a short number of nucleotides (perhaps 6 or fewer, and for functional studies, usually 1–4), and these are particularly prone to slippage and inefficient proofread- ing by DNA polymerase. The MMR system is critical to correct these problems, and if inactivated, the resulting shortening of microsatellites is a telltale sign of the ensuing ‘‘microsatellite instability’’ (MSI) phenotype. MSI testing is most often performed on mononucleotide or dinucleotide repeats.

A casual description of the MMR system would state that the MMR system is chiefly a sensory system that scans DNA, and when a nucleotide mispair is detected, removes the error and sum- mons DNA polymerase to repeat the synthesis, and this time cor- rects the error. This works because even in repetitive sequences, the error rate of DNA polymerase is low, and so simply having a second chance usually fixes the problem.

More formally, the MMR system is an excision/resynthesis sys- tem that can be divided into 4 phases: (i) recognition of a mismatch by MutS proteins, (ii) recruitment of repair enzymes, (iii) excision of the incorrect sequence, and (iv) resynthesis by DNA polymerase

using the parental strand as a template. This system is conserved through evolution from bacteria to man, with the eukaryotic system having more components that function in the recognition of mis- matches. The bacterial MMR system is biochemically the best stud- ied,

8 and the yeast and mouse systems have provided valuable

insight because of the power of genetic models. 9 However, this

review will emphasize human DNA MMR and its relevance to can- cer; there have been other recent reviews for those interested in the basic mechanisms involved in MMR.

10,11

Recognition of mismatched DNA

The human MMR protein family members responsible for rec- ognition of mispaired DNA are termed hMSH, for human MutS Homologs (of bacterial MutS which was discovered first). Human MutSa is a heterodimer composed of one molecule of hMSH2 and one molecule of hMSH6, and is the predominant form of MutS; MutSb consists of hMSH2 and hMSH3. hMSH2/6 recognizes base/base mismatches and short insertion/deletion loops whereas hMSH2/3 recognizes larger insertion/deletion loops (Figs. 1 and 2). The crystal structure of human MutS proteins has not been deter- mined yet, but the structure of bacterial MutS has given us much insight into the mechanism of mispair recognition. (Fig. 3). There is a wealth of data from biochemical studies that agrees with the structural data that MutS proteins form a sliding clamp that ini- tially binds double stranded DNA at sites of mismatches and then disengages from the mismatch to move laterally along the DNA.

12,13

hMSH proteins have one ATP binding site per molecule whose occu- pancy determines the binding properties of the MutS complex: in the presence of ADP, MutS tightly binds to mismatches, whereas in the presence of ATP, the complex acts as a sliding clamp. Lateral move- ment is necessary because at the site of a DNA mismatch there is no way for human MutS proteins to tell which strand is the correct (pa- rental) one and which strand needs to be repaired (by definition the newly synthesized one). Current models assume that the identifying feature of the newly synthesized strand is a single strand nick, e.g. gaps between Okazaki fragments in the lagging strand. This notion stems from in vitro systems that have demonstrated that MMR will excise the strand containing a nick, which is the access point that an exonuclease requires.

14

Recruitment of repair enzymes

The DNA:MutS:ATP complex recruits the MutL complex, which in humans is a heterodimer consisting of hMLH1 and hPMS2 pro- teins. MutL binds the repair complex in a mismatch-independent manner, interacting with MutS at the site of a mismatch. MutL dimers appear to function as molecular matchmakers, displacing the DNA polymerase and proliferating cell nuclear antigen (PCNA) from the nascent daughter strand, finally recruiting exonuclease I

Grant sponsor: NIH; Grant number: R01 CA72851. *Correspondence to: Department of Internal Medicine, Baylor Research

Institute, Baylor University Medical Center, H-250, BUMC, 3500 Gaston Avenue, Dallas, TX 75246, USA. Fax: 1214-818-9292. E-mail: [email protected] Received 17 November 2005; Accepted 10 March 2006 DOI 10.1002/ijc.22023 Published online 27 June 2006 in Wiley InterScience (www.interscience.

wiley.com).

Int. J. Cancer: 119, 2030–2035 (2006) ' 2006 Wiley-Liss, Inc.

Publication of the International Union Against Cancer

(Exo1) and other proteins required for ‘‘long patch excision,’’ which may excise up to a kilobase of DNA back to the site of the mismatch. Isolated hMLH1 deficiency clearly leads to MSI, as demonstrated in sporadic CRCs that arise due to epigenetic silenc- ing of hMLH1 expression.

15,16 However, mismatch-directed exci-

sion has been partially reconstituted in vitro even in the absence of MutL. Therefore, some hMLH12/2 cells may be sufficiently com- promised in MMR to exhibit MSI, but may still have residual MMR activity that can be measured using in vitro assays.

Excision of the mismatch

Exo1 has been implicated in MMR both genetically and bio- chemically. Exo12/2 mice exhibit significant deficiency in MMR of mononucleotide but not dinucleotide repeats.

17 In vitro systems demonstrate that Exo1 can mediate excision of one strand of DNA extending from a nick to a mismatch, sometimes spanning over 1,000 nucleotides, and ending about 150 nucleotides past the mis-

match. 18

Depending on the location of the nick relative to the mis- match, excision was found to proceed in either 50 > 30 or 30 > 50 directions. This appeared to be in contradiction to the exclusive 50 > 30 activity of purified Exo1, but the recent in vitro reconstitu- tion of mismatch-directed excision using purified components has demonstrated that for 50 > 30 excision, MutS, Exo1 and replication protein A (RPA) were sufficient, whereas 30 > 50 excision also

FIGURE 1 – Repair of a single nucleotide mismatch in S phase by DNA MMR (adapted from Fishel

65 ). (a) The polymerase has errone-

ously placed a G in the daughter strand across from a non-complemen- tary T in the template, creating a mismatch during S phase. (b) The heterodimer of hMSH2 and hMSH6, bound by ADP and in an open configuration, monitors newly synthesized DNA for mispairs. Upon encountering the G-T mispair, an exchange of ATP for ADP occurs, and MutSa switches to a closed, sliding clamp along the DNA. (c) The sliding clamps can migrate in either direction from the mispair, and as this occurs, additional MutSa clamps may be recruited to the mismatch. The MutSa moving in the 50 > 30 direction will eventually encounter the PCNA–DNA polymerase complex, and according to one hypothesis, displace the enzymes involved in DNA synthesis. (d) Exo1 excises the newly synthesized daughter strand back to the site of the mismatch, removing the erroneous G. (e) The error is corrected by resynthesis.

FIGURE 2 – Repair of insertion/deletion errors at microsatellite sequences. These lesions are caused by ‘‘slippage’’ during DNA repli- cation and are recognized by hMSH2 1 hMSH3 and corrected, as described in Figure 1. Mononucleotide repeats such as An (which is Tn on the complementary strand) on the left, or dinucleotide repeats (such as {CA:GT}n) on the right are recognized by hMutSb here, trig- gering ATP-ADP exchange, long-patch excision and resynthesis. For purposes of illustration, the slippage has created a short ‘‘loop out’’ on the nascent strand for the An sequence, which would lead to an insertion frameshift mutation after replication, and on the template strand for the (CA)n repeat, which would lead to a deletion mutation. This particular error is best recognized by the hMSH2 1 hMSH6 het- erodimer. The heterodimer of hMSH2 1 hMSH3 has greater affinity for larger insertion/deletion loops (not shown here) that commonly occur during DNA replication at microsatellite sequences.

FIGURE 3 – Crystal structure of prokaryotic MutS. The E. coli MutS demonstrates the N-terminal mismatch-binding domain (in blue), the ATPase domain in green (containing ADP in red), one of the mono- mers (on the left) in gray and the DNA is red, with a mismatch in yel- low. (From Sixma

66 ).

2031STRUCTURE AND FUNCTION OF THE COMPONENTS OF THE HUMAN DNA MISMATCH REPAIR SYSTEM

required MutL, PCNA and replication factor C (RFC). 19

RPA is a single strand binding protein that stabilizes the remaining strand and is required for termination of the excision once the mismatch is removed. PCNA is a homotrimeric multifunctional sliding clamp that enhances processivity of DNA polymerase, and RFC is an ATPase that loads and unloads PCNA from DNA. This differ- ential requirement for PCNA was also observed in a study that took advantage of the ability of p21

waf1 to directly inactivate

PCNA in a cell free system. p21 completely inhibited 30 excision but only partially inhibited 50 excision, suggesting that PCNA is required for 30 excision, whereas for 50 excision there are both PCNA-dependent and -independent pathways.

20 The requirement

for hMLH1 in 30 but to a lesser extent in 50 excision was also apparent in an earlier report using in vitro MMR assays in cell-free extracts.

21

DNA resynthesis

Data from bacterial and yeast systems implicated DNA poly- merase III and d, respectively, in the resynthesis step of MMR. In agreement with this, the human homolog, DNA pol d, was required for reconstitution of repair synthesis in a cell-free extract depleted of MMR activity.

24 However, because DNA pol a and e

were also present in this extract, an accessory role of these poly- merases could not be excluded. A recent study demonstrated that during somatic hypermutation of antibody genes, pol h was bound and activated by MSH2-MSH6, thus supporting earlier genetic data that showed a connection between mismatch recognition and pol h in somatic hypermutation.23

Regulation of MMR

MMR is regulated in multiple ways and responds to many stim- uli. Proliferating cells have higher levels of MMR proteins than resting cells, which makes sense because the primary function of MMR is to correct DNA replication errors, which occur primarily in growing cells. Under normal conditions in proliferating cells, the levels of individual MMR proteins relative to each other are regulated at both the mRNA and protein level.

24 This study found

that HCT116 cells, which lack hMLH1 protein because of a non- sense mutation, also lack hPMS2 protein despite the presence of hPMS2 mRNA. This could be due to either downregulation of hPMS2 translation or instability of synthesized hPMS2 protein in the absence of hMLH1. Reexpression of hMLH1 by either trans- fection or chromosome transfer restored not only hMLH1 but also hPMS2 protein. Likewise, expression of hMSH3 and hMSH6 pro- teins required the presence of hMSH2 protein. This poses a caveat to the cancer researcher or pathologist who intends to characterize the molecular defects in cancers or perform immunohistochemis- try for hPMS2 or hMSH6: absence of these proteins may indicate a defect in either of these genes or their partners, hMLH1 or hMSH2. To complicate matters further, missense mutations in the regions of protein–protein interactions among the DNA MMR members can lead to functional abrogation, even when immu- noreactivity of the protein is maintained.

25

Hypoxia due to an insufficient blood supply has been implicated as a contributing factor to genetic instability in a tumor microen- vironment. In particular, hypoxia caused enrichment of MMR-de- ficient cells in colon carcinomas and thus led to an increase in MSI in the tumors. Two recent papers found that hMLH1 as well as hMSH2 and hMSH6 were transcriptionally downregulated in hypoxic cells, and that two different mechanisms were involved. In HeLa cells, where p53 is inactivated by the papillomavirus E6 protein, hMLH1 transcription was downregulated by a mechanism that involved histone deacetylation, while hMSH2 and hMSH6 transcription remained unchanged.

26 In cells that express wild type

p53 on the other hand, hypoxia decreased transcription of hMSH2 and hMSH6 but not hMLH1.

27 This effect required the presence

of p53 and involved the displacement of the transcription factor myc from the hMSH2/6 promoters by HIF-1a. HIF-1a is a tran-

scription factor that usually induces target genes in response to hy- poxia, but in this case, caused transcriptional silencing.

A recent search for p53 target genes identified hMLH1 and hPMS2, both of which contain a putative p53 responsive element in their first intron. Upon induction of DNA damage by cisplatin, both transcripts were upregulated and p53 was found in a complex with chromosomal DNA containing the first introns of hMLH1 and hPMS2.

28

MMR downregulation was also linked to low-level microsatel- lite instability (MSI-L) under conditions of oxidative stress, partic- ularly in the setting of inflammation. Under these conditions, MMR was downregulated by a mechanism that remains elusive but was not related to transcription.

24,29,30

Bcl-2 is an anti-apoptotic protein that is overexpressed in some cancers. Bcl-2 has also been associated with increased mutability, providing a potential alternate pathway to carcinogenesis. A recent paper demonstrated that expression of Bcl-2 led to transcriptional downregulation of hMSH2 and concomitant loss of MMR activ- ity.

31 This happened through inhibition of the Cdk2-pRb-E2F

pathway; Bcl-2 directly bound and inhibited Cdk2, leading to hy- pophosphorylation of pRb, which in turn bound and inactivated the transcription factor E2F-1. Several E2F sites are present in the hMSH2 promoter, which is thus silenced in response to Bcl-2 overexpression. In addition, Bcl-2 caused higher expression of the Cdk inhibitor p27Kip1 and downregulation of cyclin E, which could contribute to inhibition of Cdk2 by Bcl-2. Taken together, it appears that Bcl-2 can have a carcinogenic effect in two ways: by inhibiting apoptosis, and by increased mutability through down- regulation of MMR.

Intracellular localization of MMR proteins is crucial as they need to be in the nucleus to repair DNA. In HeLa cells, hMLH1 and hPMS2 display nuclear localization,

32 whereas in primary

nontransformed human cells, hMLH1 is nuclear but hPMS2 is present in both the nucleus and the cytoplasm.

33 In response to

DNA damage, hPMS2 relocalizes to the nucleus. This relocaliza- tion occurs only in cells that express hMLH1 and depends upon an interaction between hPMS2 with hMLH1. Another study found that hMSH2 and hMSH6 translocate from the cytoplasm to the nu- cleus in response DNA alkylating agents.

34 Complex formation

between hMSH2 and hMSH6 appears to be important because in cells that lack hMSH6, hMSH2 fails to relocate to the nucleus. This is presumably due to the absence of a nuclear localization signal in hMSH2, which thus depends on the signal in hMSH6 to enter the nucleus. The same group found that hMSH2 and hMSH6 are phosphorylated in vitro and in vivo, and that phospho- rylation is required for nuclear translocation and G-T binding.

35,36

Phosphorylation of hMSH2 can be accomplished by PKC-f and protects hMutSa from degradation by the ubiquitin/proteasome pathway.

37

Cadmium, a known carcinogen and an environmental concern from improperly disposed rechargeable batteries, was recently shown to inhibit MMR, leading to an increase in mutation rates up to 2,000- fold.

38 In vitro assays showed that cadmium inhibits the human MutS but not MutL complexes. This is due to inhibition of the ATPase ac- tivity of hMSH6 and causes a decrease in the ability of MutS to rec- ognize mismatched DNA.

39

Signaling from MMR to cell cycle and apoptosis

The MMR system not only repairs base mismatches after DNA replication but is also involved in the processing of chemi- cal adducts in DNA caused by reagents such as alkylating agents and cisplatin (reviewed by Stojic et al.11). Methylating agents can cause two different outcomes depending on the specific moi- ety modified. Methylation of base nitrogens has no impact on base pairing and is removed by a DNA glycosylase and base excision repair, with no further consequences for the cell. Methyl- ation of base pair-forming groups, in particular O

6 in guanine,

generates modified bases that can form unusual base pairings.

2032 JASCUR AND BOLAND

O 6 -methylguanine (O

6 MeG) is normally removed by methyl-

guanine methyltransferase (MGMT), but this enzyme is silenced by promoter methylation in 40% of sporadic colon cancers as well as in other cancers. In the absence of MGMT, O

6 MeG can

pair with T instead of C in the subsequent S phase, resulting in an O

6 MeG-T mismatch. This is recognized by hMSH2/hMSH6,

and the MMR system will remove the T from the newly repli- cated strand and resynthesize that strand with a new T—repeti- tively—resulting in futile repair. It has been reported numerous times that MMR-deficient cells are relatively resistant to methy- lating agents, whereas cells with a functioning MMR system enter either G2 arrest or apoptosis, presumably depending on the severity of the DNA damage.

40,41 It was suggested that DNA

double strand breaks formed during the first replication cycle af- ter MNNG treatment were the likely intermediates that trigger apoptosis.

42 ATM, a protein kinase involved in double strand

break repair, appears to have a protective function, as ATM2/2 cells are hypersensitive to MNNG.

43 ATM also directly binds

hMLH1 and both proteins are present in the BASC complex (see later).

Cells treated with a low concentration of MNNG undergo G2 arrest that depends on the presence of hMLH1 as well as the checkpoint kinases ATR and Chk1.

44 Interestingly, the cells ar-

rested only after the second S phase following MNNG treatment, again suggesting that the signaling mechanism may require pro- cessing intermediates rather than simply recognition of O

6 MeG-T

by MutS. In addition, MMR function requires only a low level of hMLH1 protein, whereas G2 arrest following MNNG treatment requires a full dose of hMLH1, thus further arguing for separate functions of hMLH1 in MMR and signal transduction.

45

Direct binding between hMSH2 and ATR was demonstrated by Wang and Qin

46 and provides an attractive model for signaling

from the MMR machinery to the ATR cell cycle checkpoint. In this study, a higher MNNG concentration was used (10 lM vs. 0.2 lM in the previous paper) and may explain the much faster response; Chk1 phosphorylation was detected after 4–8 hr vs. 24– 48 hr. hMSH2 and Rad17 were both required for MNNG-induced G2 arrest.

Further evidence for a functional connection between MMR and ATR comes from a genetic study, which showed that ATR2/1 cells had increased chromosomal instability and sensitivity to geno- toxic stress if they were also deficient in MMR due to lack of hMLH1.

47 Mice with the same genotype (ATR2/1 MLH12/2)

had higher embryonic lethality and were prone to tumor develop- ment at earlier ages than the ATR2/1 or MLH12/2 controls. Another paper found that the stress kinase p38 was activated in

cells treated with the chemotherapeutic methylating agent TMZ and that this activation required the presence of hMLH1 and resulted in a G2 arrest. Inhibition of p38 caused cells to bypass the G2 arrest and enter mitotic catastrophes.

48

Apoptosis has been reported as a consequence of treatment of MGMT-deficient cells with DNA alkylating agents, and this out- come requires a functional MMR system, specifically MutSa.49

Apoptosis followed the mitochondrial pathway as it could be blocked by overexpression of Bcl-2. Another lab found that the apoptosis pathway triggered by MNNG depended on the cell type. Chinese hamster ovary fibroblasts followed the Bcl-2 pathway, whereas apoptosis of lymphocytes stimulated by CD3/CD28 re- ceptor engagement involved Fas/CD95.

50 The MMR system has

also been implicated in signaling from other types of DNA dam- age to apoptosis. Cisplatin-treated cells redistribute hPMS2 to the nucleus, where it binds and stabilizes the p53-related protein p73.

51 The resulting increase in p73 protein then leads to increased

apoptosis in response to cisplatin. There is, however, no absolute requirement for hPMS2 in this apoptosis pathway.

Hexavalent chromium (CrVI) is a known human carcinogen and like cadmium poses a major environmental health concern, popularized in the movie ‘‘Erin Brockovich’’. Cr(VI) forms adducts to phosphates in the DNA backbone, which trigger a cyto-

toxic response. A recent paper demonstrated that this response requires a functional MMR system.

52 Cells lacking hMLH1 or

hMSH6 show improved survival compared to wild type cells after exposure to Cr(VI). This is apparently due to DNA double strand breaks that are caused by functional MMR and trigger apoptosis. No cell cycle arrest is observed in this system.

5-fluorouracil (5-FU) is the chemotherapeutic agent most com- monly employed for CRC treatment. Unfortunately, cells defec- tive in MMR are relatively resistant to 5-FU,

53 with the result that

patients with MSI CRC do not benefit from 5-FU chemother- apy.

54,55 In vitro, 5-FU-containing DNA oligonucleotides are bound directly by purified hMSH2/hMSH6 and released by ATP.

56 While 5-FU inhibits thymidylate synthase and is primarily

incorporated into RNA, the cytotoxic effect appears to stem from 5-FU incorporation into DNA.

57 hMSH2 and hMLH1 are both

required for efficient killing by 5-FU. MMR-proficient cells incor- porate less 5-fluoro-20-deoxyuridine into DNA than MMR-defi- cient cells, indicating that this is removed from DNA by the MMR system. Finally, hMSH2/hMSH6 recognizes FU-G mispairs as effectively as T-G mispairs, and this leads to a persistent G2/M arrest.

Biochemical mechanisms of dysfunction of DNA MMR

With so many proteins involved in MMR and so many layers of regulation, it is not surprising that things can go wrong in many different ways and manifest themselves in a variety of pheno- types. Defects in MMR genes are associated with tumors in the colorectum, endometrium, stomach and many other organs. How- ever, MSI is still an unusual phenotype in most types of cancer, and when present, indicates a specific pathogenesis through DNA MMR dysfunction for those tumors. hMLH1 and hMSH2 are the most frequent targets, followed by hPMS2 and hMSH6. hPMS2 mutations have been underestimated due to the presence of at least 14 highly homologous pseudogenes that complicate genetic analysis.

58,59

Genetic alterations can either lead to absence of a protein or to a mutated protein that is functionally impaired, or it can have no consequences at all. In addition, promoter inactivation by CpG island methylation is frequently encountered with hMLH1 and is responsible for most sporadic CRCs with MSI. Diagnostic sequencing will be normal, as the lesion is epigenetic and a conse- quence of CpG sites in the hMLH1 promoter. Large genomic dele- tions encompassing several exons and introns have been found in hMSH2, resulting in no transcript and no protein being produced from the affected allele. This is less common for hMLH1. Germ- line mutations of this variety will not be found if the diagnostic strategy is restricted to exon-by-exon sequencing, since the break- points are typically intronic or in the 50 upstream sequences. Point mutations can be silent (no change in amino acid sequence), or create nonsense mutations (introducing a stop codon and resulting in a truncated protein that may be unstable) or missense mutations (changing one amino acid). In this last case, it is possible that cells generate a stable yet functionally inactive protein. The gene prod- uct will appear to be normal in the tumor by immunohistochemis- try and lead to the mistaken conclusion that this protein is not the cause of the disease, when in fact it is the culprit.

The following are a few examples of germline mutations in MMR genes that have been functionally characterized for their ability to predispose to hereditary cancer. The database of the International Society for Gastrointestinal Hereditary Tumors (InSiGHT; http://www.insight-group.org) is a valuable source for comprehensive information.

A detailed in vitro study of 7 hMSH2 mutations that cause Lynch syndrome (HNPCC) found that none affected the interac- tion between hMSH2 with hMSH6, not even those located within the interaction domain.

60 However, all of them showed reduced

binding to DNA containing a G/T mispair, and 6/7 mutants showed a reduction in ATPase activity—in one case by 1,000-

2033STRUCTURE AND FUNCTION OF THE COMPONENTS OF THE HUMAN DNA MISMATCH REPAIR SYSTEM

fold. These mutations were chosen to cover different domains of hMSH2, and one conclusion of this study was that they did not only affect the function of the domain in which they are located. Rather, one mutation in the DNA-binding domain (R524P) strongly decreased the activity of the ATP-binding domain that is located at the other end of the protein, and a mutation near the ATP-binding domain (P622L) also caused a decrease in DNA binding. This illustrates that hMSH2 is not just the sum of tethered domains, but that the different domains signal to each other to form a protein complex that functions as one unit.

61

In the case of hMLH1, Lynch syndrome mutations have a dra- matic effect on the binding to hPMS2. Of 11 mutations located in the hPMS2-binding domain of hMLH1, 9 either reduced or abol- ished the interaction with hPMS2.

62 A recent study of 34 hMLH1

mutations 25

found that 20 were expressed at low levels using a transient transfection assay, presumably due to instability of the mutated protein. Of those mutants that were expressed at high lev- els, 3 displayed decreased nuclear localization of either hMLH1 itself or hPMS2, which, as mentioned earlier, depends on hMLH1 for nuclear localization. Two others showed low activity in an in vitro MMR assay. Surprisingly, the majority of these hMLH1 mutants seemed to cause MMR dysfunction by either protein instability or mislocalization. Only two hMLH1 mutants did not

bind hPMS2, but in three cases the binding data were in conflict with previously published data.

62

A recent paper identified hMLH1 missense mutants that were defective in binding to MRE11.

63 MRE11 plays a role in double

strand break repair and is a component of the BRCA1-associated genome surveillance complex (BASC), which also contains hMSH2, hMSH6, hMLH1 and ATM.

64 In this study, downregula-

tion of MRE11 caused an increase in MSI and a decrease in MMR activity. The same region of hMLH1 that mediates interaction with hPMS2 was also required for MRE11 binding, suggesting that hPMS2 and MRE11 compete for binding MLH1, although this was not specifically shown. Of 7 mutant hMLH1 proteins, 3 bound both MRE11 and hPMS2, and 2 were defective in binding to either protein. Interestingly, 2 hMLH1 mutants were deficient in MRE11 binding but had intermediate affinity for hPMS2. The function of MRE11 in MMR is not yet known, but MRE11 has nu- clease activity and might function as an alternate nuclease to Exo1. Alternatively, MRE11 might be involved in signaling between MMR and double strand break repair. Most intriguingly, this example illustrates that mutations in MMR genes may not simply affect the basic functioning of the MMR machinery but may abrogate the interplay of MMR with other DNA repair sys- tems, or with components of cell cycle and apoptosis pathways.

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2035STRUCTURE AND FUNCTION OF THE COMPONENTS OF THE HUMAN DNA MISMATCH REPAIR SYSTEM