Báo cáo khoa học: DNA mismatch repair system Classical and fresh roles potx

MINIREVIEW DNA mismatch repair system Classical and fresh roles Sung-Hoon Jun, Tae Gyun Kim and Changill Ban Department of Chemistry and Division of Molecular & Life Science, Pohang University of Science and Technology, Korea The mismatch repair (MMR) system is essential to all organisms because it maintains the stability of the gen- ome during repeated duplication. It is composed of a few well-conserved proteins whose functions in the postreplicative repair of mismatched DNA have been characterized by co-ordinated genetic, biochemical and structural approaches. Various functions, in addition to mismatch repair during replication, have been reported for MMR proteins such as antirecombination activity between divergent sequences, promotion of meiotic crossover, DNA damage surveillance and diversification of immunoglobulins (Fig. 1). Recent research has provided a great deal of information about how MMR proteins are involved in these diverse processes. Prokaryotic mismatch repair Essential components of the MMR system – MutS, MutL, MutH and Uvr – were identified in Escherichia coli through the genetic studies of mutants that showed elevated mutation levels [1,2]. MMR reactions have also been reconstituted with purified components in E. coli [3], which drove extensive studies on prokaryotic MMR systems. MutS detects mismatches in DNA duplexes and initi- ates the MMR machinery. A microscopic study sugges- ted a possible mechanism for how MutS discriminates between heteroduplex and homoduplex DNA [4]. According to this proposal, nonspecifically bound MutS bends DNA to search for a mismatch. If it recognizes a Key words antibody diversification; DNA damage response; DNA mismatch repair; MutL; MutS Correspondence C. Ban, Department of Chemistry, Pohang University of Science and Technology, Pohang 790–784, Korea Fax: +82 54 2793399 Tel: +82 54 2792127 E-mail: ciban@postech.ac.kr (Received 12 December 2005, accepted 10 February 2006) doi:10.1111/j.1742-4658.2006.05190.x The molecular mechanisms of the DNA mismatch repair (MMR) system have been uncovered over the last decade, especially in prokaryotes. The results obtained for prokaryotic MMR proteins have provided a frame- work for the study of the MMR system in eukaryotic organisms, such as yeast, mouse and human, because the functions of MMR proteins have been conserved during evolution from bacteria to humans. However, muta- tions in eukaryotic MMR genes result in pleiotropic phenotypes in addition to MMR defects, suggesting that eukaryotic MMR proteins have evolved to gain more diverse and specific roles in multicellular organisms. Here, we summarize recent advances in the understanding of both prokaryotic and eukaryotic MMR systems and describe various new functions of MMR proteins that have been intensively researched during the last few years, including DNA damage surveillance and diversification of antibodies. Abbreviations AID, activation-induced cytidine deaminase; ATM, ataxia telangiectasia mutated; ATR, ATM and Rad3-related; Chk1, checkpoint kinase 1; Chk2, checkpoint kinase 2; CSR, class switch recombination; LC20, MutL C-terminal 20 kDa; LN40, MutL N-terminal 40 kDa; MLH, MutL homolog; MMR, mismatch repair; MSH, MutS homolog; PCNA, proliferating cell nuclear antigen; PMS, postmeiotic segregation; RPA, replication protein A; RFC, replication factor C; S, switch; SHM, somatic hypermutation; V, variable. FEBS Journal 273 (2006) 1609–1619 ª 2006 The Authors Journal compilation ª 2006 FEBS 1609 specific mismatch, MutS undergoes a conformational change and unbends the bent DNA. Crystallographic studies of Thermus aquaticus and E. coli MutS complexed with mismatched DNA provided the molecu- lar details of mismatch recognition [5–7], suggesting that a homodimer of MutS binds asymmetrically to hetero- duplex DNA (Fig. 2A). MutS has two functional domains (a DNA-binding domain and an ATPase ⁄ dimerization domain) and the asymmetry in the ATPase ⁄ dimerization domain was also reported to be essential in the MMR process in vivo [8]. These two domains are widely separated from each other, but affect each other by conformational changes that are induced by the binding of DNA or ATP [9]. This inter- action is a key molecular mechanism for modulating the function of the MutS protein in the MMR process. Only two residues, both in the same subunit of MutS, take part in the sequence-specific interaction with a mis- matched base. One, a conserved glutamate (Glu41 in T. aquaticus MutS and Glu38 in E. coli MutS), forms a hydrogen bond with the mismatched base. Recently, this hydrogen bond was suggested to induce an inhibition of the ATPase activity of MutS, helping to form a stable MutS–ATP–DNA intermediate of the downstream repair process [10]. The other specific interaction is between an aromatic ring stack of a conserved phenyl- alanine (Phe39 in T. aquaticus MutS and Phe36 in E. coli MutS) and the mismatched base. In contrast to these sequence–specific interactions, van der Waals interactions and hydrogen bonds between the DNA backbone and side chains of MutS are sequence inde- pendent [5,6]. After the recognition of mismatched DNA, MutS initiates the MMR system through direct or indirect interactions with other proteins, including MutL, MutH and UvrD. Although an exact answer to this puzzle is yet to be found, a few groups have sugges- ted various models for detailed molecular events during MMR reactions (Fig. 3) [11]. The function of MutL in the MMR system is to make a connection between the recognition of a mis- match and the excision of the mismatch from the strand within which it is contained [12]. To do this, a MutL homodimer interacts with MutS [13] and stimu- lates the endonuclease activity of MutH [14]. MutL also loads UvrD onto the DNA. UvrD is a DNA helicase II that unwinds the DNA duplex from the nick generated by MutH [15,16]. MutL is a member of the GHKL superfamily of ATPases, which includes gy- rase, a type II topoisomerase, Hsp90, histidine kinase and MutL [17]. A biochemical study demonstrated that MutL has ATPase activity [17,18]. Crystallographic studies have demonstrated that ATP binding drives dimerization of the N-terminal domain of the protein (Fig. 2B) [18], and the accompanying structural chan- ges may play key roles in co-ordinating the initial steps of mismatch recognition with downstream processing steps. A model for the intact MutL protein, which includes a large central cavity, was suggested based on Fig. 1. Various functions of mismatch repair (MMR) proteins. MMR proteins are involved in diverse genetic pathways through interac- tions with different proteins. MMR proteins increase replication fidelity by repairing errors generated during replication. Prolifer- ating cell nuclear antigen (PCNA) and replica- tion factor C (RFC) work with MMR proteins during mismatch repair in replication. Various kinds of DNA damage trigger MMR protein- dependent DNA damage responses that are implemented through the activation of ataxia telangiectasia mutated and Rad3-relat- ed (ATR) and p53. Antibody diversification is formed by mutations in immunoglobulin genes that are introduced by MMR proteins in conjunction with activation-induced cyti- dine deaminase (AID) and DNA polymerase g. In addition, MMR proteins regulate recom- bination and promote meiotic crossover. The functions of MMR proteins in green boxes are discussed in this article, whereas those in red boxes are not. Various functions of mismatch repair proteins S H. Jun et al. 1610 FEBS Journal 273 (2006) 1609–1619 ª 2006 The Authors Journal compilation ª 2006 FEBS the structures of the N-terminal domain (LN40) and the C-terminal domain (LC20), which were reported separately [17–19]. A biochemical assay, using various mutant MutL proteins, suggested that LC20 is involved in the DNA-binding activity of MutL. An increase in the DNA-binding activity of MutL also resulted in higher UvrD helicase activity [19]. MutH is a member of the type II family of res- triction endonucleases and cleaves at hemimethyl- ated GATC sites for excision of mismatch-containing strands [20]. The nicking activity of MutH is stimula- ted in a mismatch-dependent manner by MutS, MutL and ATP [20]. A structural study suggested that the C-terminal helix of MutH might act as a molecular lever through which MutS and MutL communicate and activate MutH (Fig. 2C) [21]. The nick generated by MutH serves as a point of entry for single-stranded DNA-binding protein and UvrD ⁄ helicase II, whose loading at the nick is facilitated via protein–protein interactions with MutL [15,16]. Excision of the newly synthesized strand between the nick and the mismatch is carried out by four redundant single-strand DNA- specific exonucleases: the 3¢fi5¢ exonucleases ExoI and ExoX and the 5¢fi3¢ exonucleases RecJ and ExoVII [22]. DNA polymerase III, single-stranded DNA-binding protein and DNA ligase carry out repair synthesis [3]. Eukaryotic mismatch repair All eukaryotic organisms, including yeast, mouse and human, have MutS homologs (MSHs) and MutL homologs (MLHs). The eukaryotic MMR system has been well conserved during the evolutionary process [3,23]. However, in contrast to MutS and MutL in bacteria, which function as homodimers, in eukaryotes MSHs and MLHs form heterodimers with multiple proteins. Five highly conserved MSHs (MSH2 to MSH6) are present in both yeast and mammals. MSH1, which is present in mitochondria, exists only in Fig. 2. Structures of MutS, MutL and MutH. (A) Crystal structure of the Thermus aquaticus MutS heteroduplex DNA complex (PDB acces- sion code: 1EWQ). The MutS homodimer is formed by asymmetric subunits that are represented by ribbon diagrams in green and purple. The heteroduplex DNA is a space-filling model. Two adjacent large channels with dimensions of  30 · 20 A ˚ and  40 · 20 A ˚ penetrate the disk-like protein structure, and the latter is occupied by the heteroduplex DNA. The DNA is kinked sharply towards the major groove by  60° at the unpaired base. Only one subunit (in purple) interacts with the unpaired base, thereby breaking the molecular twofold symmetry of the homodimer. (B) Crystal structure of the N-terminal 40 kDa fragment (LN40) of Escherichia coli MutL complexed with ADPnP (PDB accession code: 1B63). The structure of LN40 is homologous to that of an ATPase-containing fragment of DNA gyrase. ADPnP drives the dimerization of LN40, and the dimer interface is well ordered and made entirely of the segments that were disordered in the apoprotein. (C) A crystal structure of MutH (PDB accession code: 1AZO). The structure resembles a clamp, with a large cleft dividing the molecule into two halves. Each half forms a subdomain that contains similar structural elements. The two subdomains share a hydrophobic interface and are connected by three polypeptide linkers. The active site is located at an interface between two subdomains, and DNA binds in the cleft that is 15–18 A ˚ wide and 12–14 A ˚ deep. S H. Jun et al. Various functions of mismatch repair proteins FEBS Journal 273 (2006) 1609–1619 ª 2006 The Authors Journal compilation ª 2006 FEBS 1611 yeast [24]. MSH4 and MSH5 show reproductive tis- sue-specific expression, and null mutations of these genes do not confer mutator phenotypes because they are involved in meiotic recombination but not postrep- lication repair [25]. Genetic and biochemical studies have indicated that MSH2 is required for all mismatch correction in nuclear DNA, whereas MSH3 and MSH6 are required for the repair of some distinct and overlapping types of mismatched DNA during replica- tion [26]. These three MutS homologs make two heterodimers: MutSa (MSH2 ⁄ MSH6) and MutSb (MSH2 ⁄ MSH3). The former plays the major role in recognition of mismatched DNA in eukaryotic MMR. That is, MutSa functions in the repair of base–base mispairs as well as a range of insertion ⁄ deletion loop mispairs, whereas MutSb primarily functions in the repair of insertion ⁄ deletion loop mispairs [27,28]. MutL homologs in eukaryotic organisms were iden- tified as genes whose amino acid sequences showed high similarity with prokaryotic MutL proteins, or whose mutation phenotypes were increased levels of postmeiotic segregation (PMS) that resulted from a failure to repair mismatches in meiotic recombination intermediates [29]. There are four homologs of MutL in both yeast and mammals. In a genetic analysis, defects in MLH1 and PMS1 in yeast resulted in more severe mutator phenotypes, reminiscent of those of MSH2 and MSH6, than defects in the two other MutL homologs [30]. Also, MLH1 interacted with the other three MutL homologs in a yeast two-hybrid analysis [31]. Overall, yeast MLH1 ⁄ PMS1 and mammalian MLH1 ⁄ PMS2 heterodimers (each known as MutLa) play a major role in mutation avoidance, and the other two heterodimers of MutL homologs take part in the repair of specific classes of mismatches [32]. The bio- chemical activities and structure of MutL homologs are closely related to those of prokaryotic MutL pro- teins, especially in the N-terminal domain. The X-ray crystallographic structure of the conserved N-terminal 40-kDa fragment of human PMS2 resembles that of the ATPase fragment of E. coli MutL [33]. Extensive genetic studies in yeast have failed to find orthologs of MutH and UvrD in the MMR system, and there may be no homolog of these two proteins in the eukaryotic genome [34]. Therefore, some diver- gence in the MMR system from strand discrimination and the nicking process might occur between prokary- otes and eukaryotes. A recent increase in our know- ledge of the eukaryotic MMR system provides some understanding of this divergence. In mammalian cell extracts, mismatches provoke ini- tiation of excision at pre-existing nicks in exogenous DNA substrates with high efficiency and specificity [35,36]. The molecular nature of eukaryotic MMR could be assessed using cell extract assays in vitro, and components of the eukaryotic MMR system have been identified with depletion and complementation assays using cell extracts. One protein, identified in this way, is proliferating cell nuclear antigen (PCNA). PCNA is known to function as a processivity factor for replica- tive polymerase, but some mutations in the PCNA gene result in mutator phenotypes [37], and its interac- tions with MSH2 and MLH1 [38], and with MSH6 [39], suggest that it functions in MMR. PCNA has biochemical activity that increases the binding of MutSa to mismatched DNA; the interactions between PCNA and MSH6 are essential for this biochemical activity, which suggests that PCNA might play a role in MMR at the mispair recognition stage [39]. PCNA has been proposed to function in the mismatch recog- nition stage of MMR by helping MutSa search for mismatched DNA [40] or increasing the mismatch- binding specificity of MutSa [39]. One intriguing point about the role of PCNA in eukaryotic MMR is that the requirement for PCNA depends on the direction of the nick in the in vitro MMR assay. Although PCNA is required for mismatch-provoked excision directed by a 3¢ strand break in HeLa nuclear extracts, it is not essential for excision directed by a 5¢ nick [41,42]. Moreover, whereas 3¢ nick-directed excision is Fig. 3. Models for the assembly of the DNA mismatch repair complex in a schematic drawing. A mismatch base is detected by MutS, and ATP-bound MutS recruits MutL. In model I, the MutS–MutL complex stays at the mismatch site and activates MutH at some distance. MutS leaves the mismatch site, after bind- ing ATP, in both model II and model III. ATP is used as an energy source for translocation of MutS in model II (translocation model) but it acts as a molecular switch of MutS in model III, like GTP of G-proteins (molecular switch model). Various functions of mismatch repair proteins S H. Jun et al. 1612 FEBS Journal 273 (2006) 1609–1619 ª 2006 The Authors Journal compilation ª 2006 FEBS completely abolished by the inhibition of PCNA, 5¢ nick-directed excision is affected only minimally [42]. Finally, a mismatch-provoked 5¢fi3¢excision reaction can be reconstituted in a purified system that compri- ses only MutSa, MutLa, ExoI and replication protein A (RPA), without PCNA, and the process is similar to that observed in nuclear extracts [41]. RPA, the eukaryotic single-stranded DNA-binding protein, has been shown to enhance excision and stabilize excision intermediates in crude fractions [43,44]. The activities of ExoI are described below. Genetic studies in yeast, and biochemical studies of MMR activity in cell extracts, indicate that eukaryotes use a mechanism similar to prokaryotes, with both 3¢fi5¢ and 5¢fi3¢ exonuclease activities for mis- match correction [45]. ExoI, a 5¢fi3¢ exonuclease, was found to play a role in mutation avoidance and mismatch repair in yeast [46], and its physical inter- action with MSH2 and MLH1 also support a role in MMR [47]. Intriguingly, the mammalian ExoI was reported to be involved in both 5¢- and 3¢ nick-directed excision in extracts of mammalian cells [48], but how ExoI can have a 3¢fi5¢ exonuclease activity was unclear. Recent research by the Modrich group pro- vides a plausible answer to this question [49]. They reconstituted mismatch-provoked excision, directed by a strand break located either 3¢ or 5¢ to the mispair, in a defined human system using purified human proteins. In the presence of the eukaryotic clamp loader replica- tion factor C (RFC) and PCNA, 3¢fi5¢ excision was supported by MutSa, MutLa, ExoI and RPA. More- over, RFC and PCNA act to suppress 5¢fi3¢ excision when the strand break that directs hydrolysis is located 3¢ to the mismatch, which suggests that the polarity of mismatch-provoked excision by ExoI is regulated by PCNA and RFC. Once the strand is excised beyond the mismatch, DNA resynthesis occurs by the activity of polymerase d [50] in the presence of PCNA [51] and RPA [43,44]. The remaining nick is then sealed by an as-yet-unidentified ligase, completing the repair process. MMR proteins in the DNA damage response The involvement of MMR proteins in the DNA dam- age response first became apparent when it was discov- ered that MMR-defective bacterial and mammalian cells are resistant to cell death caused by alkylating agents [52]. MMR-deficient cells are also resistant to other DNA-damaging agents, including methylation agents, cisplatin and UV radiation [53]. Subsequent studies on the roles of MMR proteins in response to DNA damage in normal cells showed that N-methyl- N¢-nitro-N-nitrosoguanidine, an alkylating agent, trig- gers MMR-dependent G2 ⁄ M arrest [54], which is followed by the induction of MMR-dependent apopto- sis [55]. The role of MMR proteins in response to DNA damage can be inferred from the interactions of MMR proteins with the tumor suppressor protein, p53, and p53-related proteins. p53 acts as a major point in a complex network that responds to diverse cellular stresses, including DNA damage [56]. Once stabilized and activated by genotoxic stress, p53 can either acti- vate or repress a wide array of different gene targets by binding to their promoter regions, which in turn can regulate cell cycle, cell death and other outcomes [57]. The p53 homologs p63 and p73 induce p53-inde- pendent apoptosis as well as affect trans-activation of certain target genes by p53 [58,59]. Treatment of human cells with methylating agents results in phos- phorylation of p53 and induction of apoptosis, a response that depends on the presence of functional hMutSa and hMutLa [60]. UVB-induced apoptosis is significantly reduced in MSH2-deficient cells, and it correlates with decreased activation of p53, which sug- gests that MSH2 may act upstream of p53 to induce post-UVB apoptosis [61]. Cisplatin-caused DNA dam- age increases the stability of p73, which induces apop- tosis that is dependent on functional hMLH1 protein [62]. Moreover, cisplatin stimulates the interaction between PMS2 and p73, which is required for the acti- vation of p73 and subsequent induction of apoptosis [63]. PMS2 and p73 can also interact with each other, independently of MLH1, suggesting that MMR pro- teins have specific roles in the DNA damage response. Taken together, these reports indicate that MMR pro- teins may play roles in multiple steps of the DNA damage response, as damage sensors and adaptors of the pathways (Fig. 4). The roles of MMR proteins in the response to DNA damage are further supported by the failure of MMR mutants to trigger G2 ⁄ M arrest in response to the methylator N-methyl-N¢-nitro-N-nitrosoguanidine and similar alkylators [64]. The G2 ⁄ M checkpoint prevents cells from initiating mitosis when they experience DNA damage during G2, or when they progress into G2 with unrepaired damage incurred during the previ- ous S or G1 phases [65]. A study with a cell line lack- ing hMLH1 expression and an inducible hMLH1 expression system showed that methylation-induced G2 ⁄ M arrest requires a full complement of hMLH1 (expression level similar to that of the wild type), whereas MMR proficiency was restored, even at low hMLH1 concentrations ( 10% of wild-type expres- S H. Jun et al. Various functions of mismatch repair proteins FEBS Journal 273 (2006) 1609–1619 ª 2006 The Authors Journal compilation ª 2006 FEBS 1613 sion) [66], suggesting that these two responses are carried out by different genetic pathways. The compo- nents that transduce the G2 ⁄ M checkpoint signal pathway, such as ataxia telangiectasia mutated (ATM), ATM and Rad3-related (ATR), checkpoint kinase 1 (Chk1) and checkpoint kinase 2 (Chk2), are activated in the MMR system-dependent G2 ⁄ M arrest induced by DNA methylation [67,68]. ATR and Chk1 path- ways are essential for this response [67]. The mitogen- activated protein (MAP) kinase, p38a, is activated in MMR-proficient cells exposed to the methylating agent, temozolomide, but not in MLH1 knockdown cells [69], suggesting that the p38 MAP kinase pathway links the MMR system to the G2 ⁄ M checkpoint. The interaction between MMR proteins and checkpoint proteins also suggests direct roles for MMR proteins in the DNA damage response. Both in vitro and in vivo approaches show that MSH2 binds to Chk1 and Chk2 [68], that MLH1 associates with ATM [70] and that these interactions are enhanced after treatment with a methylating agent [68]. MSH2 protein physically inter- acts with ATR in the damage response to DNA methylation, and their interaction is required for the phosphorylation of Chk1 [71]. ATR also serves as a haploinsufficient tumor suppressor in MMR-deficient cells, suggesting the genetic interaction of these pro- teins [72]. Taken together, these findings suggest that MMR proteins function early in the pathway that leads from DNA methylating agents to G2⁄ M arrest (Fig. 4). The molecular mechanism of the involvement of MMR proteins in various DNA damage responses is unclear. Given the original function of the MMR system in detecting and repairing errors that occur during replication, the MMR protein complex could serve as a sensor for DNA damage [71]. A large com- plex, named BRCA1-associated genome surveillance complex, which includes tumor suppressors and the MMR ⁄ DNA damage-repair proteins MSH2, MSH6, MLH1, ATM, Bloom’s syndrome, and RAD50– MRE11–NBS1, has also been suggested to be a poss- ible sensor for DNA damage [73]. The roles of MMR proteins in the DNA damage response may not be simple from the viewpoint of their various relation- ships with other regulators of the DNA damage response, especially with ATM and p53. For instance, hMLH1 and hPMS2 were identified as direct target genes of p53 [74]. Cisplatin induces the accumulation of hPMS1, hPMS2 and hMLH1 through ATM-medi- ated protein stabilization, and the induced level of these MMR proteins is important for the phosphorylat- ion of p53 by ATM in the response to DNA damage [75]. MMR proteins and p53 therefore may act as a kind of positive feedback regulation for the DNA damage response, or a more complicated network may regulate their activity and expression. MMR proteins in antibody diversification In addition to the initial generation of antibody diver- sity by gene rearrangement during B-cell development [76], specific antigen recognition triggers a second wave of antibody diversification through somatic hypermu- tation (SHM) and class switch recombination (CSR). SHM introduces multiple single-nucleotide substitut- ions into variable (V) regions of immunoglobulin Fig. 4. A simplified model of DNA damage response pathways that are dependent on mismatch repair (MMR) proteins. MMR proteins bind to damaged DNA and recruit various signal-transducing kinases, including ataxia telangiectasia mutated (ATM), ATM and Rad3-related (ATR), and checkpoint kinase 1 ⁄ checkpoint kinase 2 (Chk1 ⁄ Chk2). They in turn stabilize and activate p53, a key compo- nent in DNA damage responses, such as cell cycle checkpoint activation and programmed cell death (apoptosis). p73, a p53 homolog, is also a transducer of the MMR protein-dependent DNA damage response, and postmeiotic segregation 2 (PMS2) is known to bind and stabilize p73. The p38 mitogen-activated protein (MAP) kinase pathway connects MMR proteins and p53 ⁄ p73 in this pathway. c-Abl is a tyrosine kinase that acts upstream of p73 and stabilizes it [59]. Various functions of mismatch repair proteins S H. Jun et al. 1614 FEBS Journal 273 (2006) 1609–1619 ª 2006 The Authors Journal compilation ª 2006 FEBS genes and CSR is a region-specific intrachromosomal recombination that replaces the Cl form of the immu- noglobulin (Ig) heavy chain constant region (C H ) gene with other C H genes, resulting in a switch of the Ig iso- type from IgM to IgG, IgE, or IgA [77]. The mole- cular processes of SHM and CSR, and the proteins involved in these processes, have been investigated in detail over the last few years. Advances in gene target- ing techniques have led to the availability of mice with loss-of-function mutations in MMR genes, and recent studies using these mice have suggested that MMR proteins are directly involved in antibody diversifica- tion. MSH2-deficient mice accumulated fivefold fewer mutations in the V region of antibody genes [78]. MSH6 deficiency caused similar effects, but MSH3 deficiency did not [79], suggesting that MutSa plays an essential role in SHM. Similarly, mice with loss- of-function mutations in MSH2 or MSH6 have a decreased frequency of CSR, but those with MSH3 do not [80]. Mice carrying a mutation in the MSH2 ATPase domain are deficient in SHM and CSR, sug- gesting that the ATPase activity of MSH2 is essential for antibody diversification [81]. It will be interesting to understand how MMR proteins are involved in the processes of SHM and CSR, which require the induc- tion of mutations. Both SHM and CSR start with the activity of acti- vation-induced cytidine deaminase (AID), which is a homolog of the RNA editing enzyme, but is known to deaminate dC to dU subsequently in ssDNA [82,83]. Transcription of the Ig gene in the V and switch (S) regions is required for SHM and CSR, respectively [84], because AID deaminates cytidine residues in single-stranded DNA located in the tran- scription bubble of the V and S regions [85,86], (Fig. 5). AID-induced mutations of cytidine explain some SHM and mutations in CSR, but up to half of the mutations of the V and S regions are independent of AID. Phenotypic analyses of MSH2- and MSH6- defective mice showed that the spectra of SHM were different in these mice than in wild-type mice [78,79], indicating that MSH2 and MSH6 are required for mutations at AT base pairs during SHM and CSR [78,79]. These results suggested that mutations in SHM and CSR are achieved in two steps: in the first step, AID generates mutations in GC base pairs, and in the second step, the MMR system is recruited to the mismatched DNA and resynthesizes the DNA strand with the help of an error-prone polymerase, such as pol g (Fig. 5) [87]. This model is supported by a report that MSH a not only binds to a U:G mispair, but also physically interacts with DNA poly- merase g and functionally stimulates its catalytic activity [88]. Moreover, the phenotypes of mice mutant for ExoI are similar to those of MSH2– ⁄ – mice, with reduced SHM and CSR, and ExoI and MLH1 physically interact with mutating variable regions [89]. A B C D Fig. 5. A model of somatic hypermutation that is dependent on the mismatch repair (MMR) protein. (A) During transcription of the immuno- globulin gene in the variable (V) region, activation-induced cytidine deaminase (AID) deaminates cytidine residues in single-stranded DNA to produce UG mismatches. (B) MutSa and MutLa are recruited to the mismatched DNA, and activate ExoI. (C) The gaps generated by the activity of ExoI are refilled by error-prone DNA polymerase g, resulting in mutations in AT base pairs. (D) The diversity of the V regions of antibody genes is thus accomplished by the formation of mutations by a mechanism that depends on MMR proteins. S H. Jun et al. Various functions of mismatch repair proteins FEBS Journal 273 (2006) 1609–1619 ª 2006 The Authors Journal compilation ª 2006 FEBS 1615 Conclusion The MMR system was originally discovered as a mechanism that maintains the integrity of the genome during replication. Increasingly, however, components of the system are being found to participate in diverse cellular processes, including the repair of DNA dam- age and antibody diversification. How MMR proteins are regulated to perform these various functions will be an important question for the co-ordinated under- standing of MMR proteins. Searching for the unidenti- fied components of the MMR system will also provide further information in the growing body of research on the mechanism of the MMR system. Finally, understanding the MMR system will provide insights into cancer development related to the defects in MMR genes and the treatment of tumors, both hered- itary and sporadic, with defective MMR. Acknowledgements This work was supported by the Center for Integrated Molecular Systems through KOSEF, the POSRIP res- earch grant (1RC0402301), and the Center for Innova- tive Bio-Physio Technology at BNU (grant number: 02-PJ3-PG6-EV05-0001). References 1 Wagner R & Meselson M (1976) Repair tracts in mis- matched DNA heteroduplexes. Proc Natl Acad Sci USA 73, 4135–4139. 2 Cox EC, Degnen GE & Scheppe ML (1972) Mutator gene studies in Escherichia coli: the mutS gene. Genetics 72, 551–567. 3 Modrich P & Lahue R (1996) Mismatch repair in repli- cation fidelity, genetic recombination, and cancer biol- ogy. Annu Rev Biochem 65, 101–133. 4 Wang H, Yang Y, Du Schofield MJC, Fridman Y, Lee SD & Larson ED (2003) DNA bending and unbending by MutS govern mismatch recognition and specificity. Proc Natl Acad Sci USA 100, 14822–14827. 5 Obmolova G, Ban C, Hsieh P & Yang W (2000) Crystal structures of mismatch repair protein MutS and its complex with a substrate DNA. Nature 407, 703–710. 6 Lamers MH, Perrakis A, Enzlin JH, Winterwerp HH, de Wind N & Sixma TK (2000) The crystal structure of DNA mismatch repair protein MutS binding to a GT mismatch. Nature 407, 711–717. 7 Junop MS, Obmolova G, Rausch K, Hsieh P & Yang W (2001) Composite active site of an ABC ATPase: MutS uses ATP to verify mismatch recognition and authorize DNA repair. Mol Cell 7, 1–12. 8 Lamers MH, Winterwerp HH & Sixma TK (2003) The alternating ATPase domains of MutS control DNA mis- match repair. EMBO J 22, 746–756. 9 Lamers MH, Georgijevic D, Lebbink JH, Winterwerp HH, Agianian B, de Wind N & Sixma TK (2004) ATP increases the affinity between MutS ATPase domains. J Biol Chem 279, 43879–43885. 10 Lebbink JH, Georgijevic D, Natrajan G, Fish A, Winter- werp HH, Sixma TK & de Wind N (2006) Dual role of MutS glutamate 38 in DNA mismatch discrimination and in the authorization of repair. EMBO J 25, 409–419. 11 Kunkel TA & Erie DA (2005) DNA mismatch repair. Annu Rev Biochem 74, 681–710. 12 Sancar A & Hearst JE (1993) Molecular matchmakers. Science 259, 1415–1420. 13 Galio L, Bouquet C & Brooks P (1999) ATP hydro- lysis-dependent formation of a dynamic ternary nucleo- protein complex with MutS and MutL. Nucleic Acids Res 27, 2325–2331. 14 Hall MC & Matson SW (1999) The Escherichia coli MutL protein physically interacts with MutH and sti- mulates the MutH-associated endonuclease activity. J Biol Chem 274, 1306–1312. 15 Dao V & Modrich P (1998) Mismatch-, MutS-, MutL-, and helicase II-dependent unwinding from the single- strand break of an incised heteroduplex. J Biol Chem 273, 9202–9207. 16 Hall MC, Jordan JR & Matson SW (1998) Evidence for a physical interaction between the Escherichia coli methyl-directed mismatch repair proteins MutL and UvrD. EMBO J 17, 1535–1541. 17 Ban C & Yang W (1998) Crystal structure and ATPase activity of MutL: implications for DNA repair and mutagenesis. Cell 95, 541–552. 18 Ban C, Junop M & Yang W (1999) Transformation of MutL by ATP binding and hydrolysis: a switch in DNA mismatch repair. Cell 97, 85–97. 19 Guarne A, Ramon-Maiques S, Wolff EM, Ghirlando R, Hu X, Miller JH & Yang W (2004) Structure of the MutL C-terminal domain: a model of intact MutL and its roles in mismatch repair. EMBO J 23, 4134–4145. 20 Au KG, Welsh K & Modrich P (1992) Initiation of methyl-directed mismatch repair. J Biol Chem 267, 12142–12148. 21 Ban C & Yang W (1998) Structural basis for MutH activation in E. coli mismatch repair and relationship of MutH to restriction endonucleases. EMBO J 17, 1526– 1534. 22 Burdett V, Baitinger C, Viswanathan M, Lovett ST & Modrich P (2001) In vivo requirement for RecJ, ExoVII, ExoI, and ExoX in methyl-directed mismatch repair. Proc Natl Acad Sci USA 98 , 6765–6770. 23 Kolodner RD (1996) Biochemistry and genetics of euk- aryotic mismatch repair. Genes Dev 10, 1433–1442. Various functions of mismatch repair proteins S H. Jun et al. 1616 FEBS Journal 273 (2006) 1609–1619 ª 2006 The Authors Journal compilation ª 2006 FEBS 24 Reenan RAG & Kolodner RD (1992) Characterization of insertion mutations in the Saccharomyces cerevisiae MSH1 and MSH2 genes: evidence for separate mito- chondrial and nuclear functions. Genetics 132, 975–985. 25 Ross-Macdonald P & Roeder GS (1994) Mutation of a meiosis-specific MutS homolog decreases crossing over but not mismatch correction. Cell 79, 1069–1080. 26 Marsischky GT, Filosi N, Kane MF & Kolodner R (1996) Redundancy of Saccharomyces cerevisiae MSH3 and MSH6 in MSH2-dependent mismatch repair. Genes Dev 10, 407–420. 27 Genschel J, Littman SJ, Drummond JT & Modrich P (1998) Isolation of MutSb from human cells and com- parison of the mismatch repair specificities of MutSb and MutSa. J Biol Chem 273, 19895–19901. 28 Umar A, Risinger JI, Glaab WE, Tindall KR, Barrett JC & Kunkel TA (1998) Functional overlap in mis- match repair by human MSH3 and MSH6. Genetics 148, 1637–1646. 29 Kramer W, Kramer B, Williamson MS & Fogel S (1989) Cloning and nucleotide sequence of DNA mis- match repair gene PMS1 from Saccharomyces cerevi- siae: homology of PMS1 to procaryotic MutL and HexB. J Bacteriol 171, 5339–5346. 30 Prolla T, Christie DM & Liskay RM (1994) Dual requirement in yeast DNA mismatch repair for MLH1 and PMS1, two homologs of the bacterial mutL gene. Mol Cell Biol 14, 407–415. 31 Wang TF, Kleckner N & Hunter N (1999) Functional specificity of MutL homologs in yeast: Evidence for three Mlh1-based heterocomplexes with distinct roles during meiosis in recombination and mismatch correc- tion. Proc Natl Acad Sci USA 96, 13914–13919. 32 Harfe BD, Minesinger BK & Jinks-Robertson S (2000) Discrete in vivo roles for the MutL homologs Mlh2p and Mlh3p in the removal of frameshift intermediates in budding yeast. Curr Biol 10, 145–148. 33 Guarne A, Junop MS & Yang W (2001) Structure and function of the N-terminal 40 kDa fragment of human PMS2: a monomeric GHL ATPase. EMBO J 20, 5521– 5531. 34 Hafe BD & Robertson SJ (2000) DNA mismatch repair and genetic instability. Annu Rev Genet 34, 359–399. 35 Holmes J, Clark S & Modrich P (1990) Strand-specific mismatch correction in nuclear extracts of human and Drosophila melanogaster cell lines. Proc Natl Acad Sci USA 87, 5837–5841. 36 Iams K, Larson ED & Drummond JT (2002) DNA template requirements for human mismatch repair in vitro. J Biol Chem 277, 30805–30814. 37 Ayyagari R, Impellizzeri KJ, Yoder BL, Gary SL & Bergers PM (1995) A multinational analysis of the yeast proliferating cell nuclear antigen indicates distinct roles in DNA replication and DNA repair. Mol Cell Biol 15, 4420–4429. 38 Umar A, Buermeyer AB, Simon JA, Thomas DC, Clark AB, Liskay RM & Kunkel TA (1996) Requirement for PCNA in DNA mismatch repair at a step preceding DNA resynthesis. Cell 87, 65–73. 39 Flores-Rozas H, Clark D & Kolodner RD (2000) Prolif- erating cell nuclear antigen and Msh2p-Msh6p interact to form an active mispair recognition complex. Nat Genet 26, 375–378. 40 Lau PJ & Kolodner RD (2003) Trnasfer of the MSH2- MSH6 complex from proliferating cell nuclear antigen to mispaired bases in DNA. J Biol Chem 278, 14–17. 41 Genschel J & Modrich P (2003) Mechanism of 5¢ direc- ted excision in human mismatch repair. Mol Cell 12, 1077–1086. 42 Guo S, Presnell SR, Yuan F, Zhang Y, Gu L & Li GM (2004) Differential requirement for proliferating cell nuclear antigen in 5¢-and 3¢ nick-directed excision in human mismatch repair. J Biol Chem 279, 16912–16917. 43 Lin YL, Shivji MKK, Chen C, Kolodner R, Wood RD & Dutta A (1998) The evolutionarily conserved zinc fin- ger motif in the largest subunit of human replication protein A is required for DNA replication and mis- match repair but not for nucleotide excision repair. J Biol Chem 273, 1453–1461. 44 Ramilo C, Gu L, Guo S, Zhang X, Patrick SM, Turchi JJ & Li GM (2002) Partial reconstitution of human DNA mismatch repair in vitro: characterization of the role of human replication protein A. Mol Cell Biol 22, 2037–2046. 45 Tran HT, Gordenin DA & Resnick MA (1999) The 3¢fi5¢ exonucleases of DNA polymerases d and e and the 5¢fi3¢ exonuclease Exo1 have major roles in post- replication mutation avoidance in Saccharomyces cere- visiae. Mol Cell Biol 19, 2000–2007. 46 Huang KN & Symington LS (1993) A 5¢fi3¢ exonuc- lease from Saccharomyces cerevisiae is required for in vitro recombination between linear DNA molecules with overlapping homology. Mol Cell Biol 13, 3125–3134. 47 Tran PT, Simon JA & Liskay RM (2001) Interactions of Exo1p with components of MutLa in Saccharomyces cerevisiae. Proc Natl Acad Sci USA 98, 9760–9765. 48 Genschel J, Bazemore LR & Modrich P (2002) Human exonuclease I is required for 5¢- and 3¢ mismatch repair. J Biol Chem 277, 13302–13311. 49 Dzantiev L, Constantin N, Genschel J, Iyer RR, Burg- ers PMJ & Modrich P (2004) A defined human system that supports bidirectional mismatch-provoked excision. Mol Cell 15, 31–41. 50 Longley MJ, Pierce AJ & Modrich P (1997) DNA poly- merase d is required for human mismatch repair in vitro. J Biol Chem 272, 10917–10921. 51 Gu L, Hong Y, McCulloch S, Watanabe H & Li GM (1998) ATP-dependent interaction of human mismatch repair proteins and dual role of PCNA in mismatch repair. Nucleic Acids Res 26, 1173–1178. S H. Jun et al. Various functions of mismatch repair proteins FEBS Journal 273 (2006) 1609–1619 ª 2006 The Authors Journal compilation ª 2006 FEBS 1617 52 Branch P, Aquilina G, Bignami M & Karran P (1993) Defective mismatch binding and a mutator phenotype in cells tolerant to DNA damage. Nature 362, 652–654. 53 Fink D, Aebi S & Howell SB (1998) The role of DNA mismatch repair in drug resistance. Clin Cancer Res 4, 1–6. 54 Hawn MT, Umar A, Carethers JM, Marra G, Kunkel TA, Boland CR & Koi M (1995) Evidence for a connec- tion between the mismatch repair system and the G2 cell cycle checkpoint. Cancer Res 55, 3721–3725. 55 Tominaga Y, Tsuzuki T, Shiraishi A, Kawate H & Sekiguchi M (1997) Alkylation-induced apoptosis of embryonic stem cells in which the gene for DNA-repair, methyltransferase, had been disrupted by gene targeting. Carcinogenesis 18, 889–896. 56 Ko LJ & Prives C (1996) P53: puzzle and paradigm. Genes Dev 10, 1054–1072. 57 Johnstone RW, Ruefli AA & Lowe SW (2002) Apopto- sis: a link between cancer genetics and chemotherapy. Cell 108, 153–164. 58 Flores ER, Tsai KY, Crowley D, Sengupta S, Yang A, McKeon F & Jacks T (2002) P63 and p73 are required for p53-dependent apoptosis in response to DNA damage. Nature 416, 560–564. 59 Jost C, Marin M & KaelinW (1997) p73 is a human p53-related protein that can induce apoptosis. Nature 389, 191–194. 60 Duckett DR, Bronstein SM, Taya Y & Modrich P (1999) hMutSa and hMutLa-dependent phosphorylation of p53 in response to DNA methylator damage. Proc Natl Acad Sci USA 96, 12384–12388. 61 Peters AC, Young LC, Maeda T, Tron VA & Andrew SE (2003) Mammalian DNA mismatch repair protects cells from UVB-induced DNA damage by facilitating apoptosis and p53 activation. DNA Repair (Amster- dam) 2, 427–435. 62 Gong JG, Costanzo A, Yang HQ, Melino G, Kaelin WG Jr, Levrero M & Wang JY (1999) The tyrosine kinase c-Abl regulates p73 in apoptosis response to cis- platin-induced DNA damage. Nature 399, 806–809. 63 Shimodaira H, Yamashita AY, Kolodner RD & Wang JYJ (2003) Interaction of mismatch repair protein PMS2 and the p53-related transcription factor p73 in apoptosis response to cisplatin. Proc Natl Acad Sci USA 100, 2420–2425. 64 Kat A, Thilly WG, Fang WH, Longley MJ, Li GM & Modrich P (1993) An alkylation-tolerant, mutator human cell line is deficient in strandspecific mismatch repair. Proc Natl Acad Sci USA 90 , 6424–6428. 65 Nyberg KA, Michelson RJ, Putnam CW & Weinert TA (2002) Toward maintaining the genome: DNA damage and replication checkpoints. Annu Rev Genet 36, 617– 656. 66 Cejka P, Stojic L, Mojas N, Russell AM, Heinimann K, Cannavo E, di Pietro M, Marra G & Jiricny J (2003) Methylation-induced G(2) ⁄ M arrest requires a full com- plement of the mismatch repair protein hMLH1. EMBO J 22, 2245–2254. 67 Stojic L, Mojas N, Cejka P, Di Pietro M, Ferrari S, Marra G & Jiricny J (2004) Mismatch repair-dependent G2 checkpoint induced by low doses of SN1 type methylating agents requires the ATR kinase. Genes Dev 18, 1331–1344. 68 Adamson AW, Beardsley DI, Kim W, Gao Y, Baskaran R & Brown KD (2005) Methylator-induced, mismatch repair-dependent G2 arrest is activated through Chk1 and Chk2. Mol Biol Cell 16, 1513–1526. 69 Hirose Y, Katayama M, Stokoe D, Kogan DAH, Ber- ger MS & Pieper RO (2003) The p38 mitogen-activated protein kinase pathway links the DNA mismatch repair system to the G2 checkpoint and to resistance to che- motherapeutic DNA-methylating agents. Mol Cell Biol 23, 8306–8315. 70 Brown KD, Rathi A, Kamath R, Beardsley DI, Zhan Q, Mannino JL & Baskaran R (2003) The mismatch repair system is required for S-phase checkpoint activa- tion. Nat Genet 33, 80–84. 71 Wang Y & Qin J (2003) MSH2 and ATR form a signal- ing module and regulate two brnaches of the damage response to DNA methylation. Proc Natl Acad Sci USA 100, 15387–15392. 72 Fang Y, Tsao CC, Goodman BK, Furumai R, Tirado CA, Abraham RT & Wang XF (2004) ATR functions as a gene dosage-dependent tumor suppressor on a mis- match repair-deficient background. EMBO J 23, 3164– 3174. 73 Wang Y, Cortez D, Yazdi P, Neff N, Elledge SJ & Qin J (2000) BASC, a super complex of BRCA1-asso- ciated proteins involved in the recognition and repair of aberrant DNA structures. Genes Dev 14, 927–939. 74 Chen J & Sadowski I (2005) Identification of the mis- match repair genes PMS2 and MLH1 as p53 target genes by using serial analysis of binding elements. Proc Natl Acad Sci USA 102, 4813–4818. 75 Luo Y, Lin F & Lin W (2004) ATM-mediated stabiliza- tion of hMutL DNA mismatch repair proteins aug- ments p53 activation during DNA damage. Mol Cell Biol 24, 6430–6444. 76 Tonegawa S (1983) Somatic generation of antibody diversity. Nature 302, 575–581. 77 MacLennan IC (1994) Germinal centers. Annu Rev Immunol 12, 117–139. 78 Rada C, Ehrenstein MR, Neuberger MS & Milstein C (1998) Hot spot focusing of somatic hypermutation in MSH-deficient mice suggests two stages of mutational targeting. Immunity 9, 135–141. 79 Wiesendanger M, Kneitz B, Edelmann W & Scharff MD (2000) Somatic mutation in MSH3, MSH6, and MSH3 ⁄ MSH6-deficient mice reveals a role for the Various functions of mismatch repair proteins S H. Jun et al. 1618 FEBS Journal 273 (2006) 1609–1619 ª 2006 The Authors Journal compilation ª 2006 FEBS [...]... Class switch recombination and hypermutation require activation-induced cytidine deaminase (AID), a potential RNA editing enzyme Cell 102, 553–563 Pham P, Bransteitter R, Petruska J & Goodman M (2003) Processive AID-catalysed cytosine deamination on single-stranded DNA simulates somatic hypermutation Nature 424, 103–107 Storb U (1998) Progress in understanding the mechanism and consequences of somatic... Rev 162, 5–11 Various functions of mismatch repair proteins 85 Bransteitter R, Pham P, Shcarff MD & Goodman MF (2003) Activation-induced cytidine deaminase deaminates deoxycytidine on single-stranded DNA but requires the action of Rnase Proc Natl Acad Sci USA 100, 4102–4107 86 Chaudhuri J, Tian M, Khuong C, Chua K, Pinaud E & Alt FW (2003) Transcription-targeted DNA deamination by the AID antibody... Edelmann W et al (2004) Examination of Msh6- and Msh3-deficient mice in class switching reveals overlapping and distinct roles of MutS homologues in antibody diversification J Exp Med 200, 47–59 Martin A, Li Z, Lin D, Bardwell PD, Iglesias-Ussel MD, Edelmann W & Scharff MD (2003) Msh2 ATPase activity is essential for somatic hypermutation at A-T basepairs and for efficient class switch recombination J... 726–730 87 Zeng X, Winter DB, Kasmer C, Kraemer KH, Lehmann AR & Gearhart PJ (2001) DNA polymerase eta is an A-T mutator in somatic hypermutation of immunoglobulin variable genes Nat Immunol 2, 537–541 88 Wilson TM, Vaisman A, Martomo SA, Sullivan P, Lan L, Hanaoka F, Yasui A, Woodgate R & Gearhart PJ (2005) MSH2–MSH6 stimulates DNA polymerase g, suggesting a role for A: T mutations in antibody genes J Exp... suggesting a role for A: T mutations in antibody genes J Exp Med 201, 637–645 89 Bardwell PD, Woo CJ, Wei K, Li Z, Martin A, Sack SZ, Parris T, Edelmann W & Scharff MD (2004) Altered somatic hypermutation and reduced class-switch recombination in exonuclease 1-mutant mice Nat Immunol 5, 224–229 FEBS Journal 273 (2006) 1609–1619 ª 2006 The Authors Journal compilation ª 2006 FEBS 1619 . MINIREVIEW DNA mismatch repair system Classical and fresh roles Sung-Hoon Jun, Tae Gyun Kim and Changill Ban Department of Chemistry and Division of. RecJ and ExoVII [22]. DNA polymerase III, single-stranded DNA- binding protein and DNA ligase carry out repair synthesis [3]. Eukaryotic mismatch repair All