Mismatch repair

Specific repair systems are activated in response to DNA lesions. Mismatch repair protects the genome of prokaryotic and eukaryotic cells from errors arising during replication or induced by mutagenic factors. The mismatch repair system distinguishes between the newly synthesized and pattern DNA strands by the extent of methylation and checks the accuracy of genetic information after homologous recombination. Very short-patch repair corrects mismatches in CC(A/T)GG sites. The 8-oxoguanine system is independent of DNA hemimethylation and removes oxidized bases from prokaryotic and eukaryotic genomes. Mutations of repair genes increase mutagenesis in prokaryotic cells and cause colorectal cancer in humans. The review considers the repair mechanisms and the role of repair defects in mutagenesis and carcinogenesis.     

Mismatch repair genes of eukaryotes

Mismatches that arise during replication or genetic recombination or owing to damage to DNA by chemical agents are recognized by mismatch repair systems. The pathway has been characterized in detail inEscherichia coll. Several homologues of the genes encoding the proteins of this pathway have been identified in the yeastSaccharomyces cerevisiae and in human cells. Mutations in the human geneshMSH2, hMLH1, hPMS1 andhPMS2 have been linked to hereditary nonpolyposis colon cancer (HNPCC) and to some sporadic tumours. Mismatch repair also plays an antirecombinogenic role and is implicated in speciation.     

Mismatch repair and homeologous recombination

DNA mismatch repair influences the outcome of recombination events between diverging DNA sequences. Here we discuss how mismatch repair proteins are active in different homologous recombination subpathways and specific reaction steps, resulting in differential modulation of these recombination events, with a focus on the mechanism of heteroduplex rejection during the inhibition of recombination between slightly diverged (homeologous) DNA sequences.     

Mismatch repair and cancer susceptibility

Mismatch-repair systems have been identified in organisms ranging from Escherichia coli to humans. They can repair almost all DNA base pair mismatches as well as small insertion/deletion mismatches. Molecular and biochemical analyses have shown that the core components of eukaryotic mismatch-repair systems are highly homologous to their bacterial counterparts. In humans, defects in four mismatch-repair genes have been linked both to hereditary non-polyposis colorectal cancer and to spontaneous cancers that exhibit rearrangements in DNA containing simple repeat sequences.     

Mismatch repair outside of replication

Comment on: Rodriguez GP, et al. Proc Natl Acad Sci USA 2012; 109:6153-8.     

Mismatch repair in mammalian cells

A vital process in maintaining a low genetic error rate is the removal of mismatched bases in DNA. The importance of this process in E. coli is demonstrated by the 100–1000 fold increase in mutation frequency observed in cells deficient in this repair system⁽¹⁾. Mismatches can arise as a consequence of recombination, errors in replication and as a result of spontaneous chemical deamination, the latter process resulting in an estimated twelve T:G mismatches per genome per day in mammalian cells⁽²⁾. Recent studies, discussed here, provide evidence for the existence of specific mismatch repair systems in mammalian and human cells.     

Mismatch repair defects in cancer

Post-replicative mismatch repair in humans utilises the hMSH2, hMSH6, hMSH3, hMLH1 and hPMS2 genes and possibly the newly identified hMLH3 gene. Recently, a link has been established between hMSH6 mutations and 'atypical' hereditary non-polyposis colon cancer (HNPCC) with an increased incidence of endometrial cancers. To satisfy the need for a diagnostic test capable of differentiating between pathogenic mutations and polymorphisms, several functional assays that fulfil these criteria have been described. These should allow for better diagnosis of HNPCC.     

Mismatch repair outside of replication

Comment on: Rodriguez GP, et al. Proc Natl Acad Sci USA 2012; 109:6153-8.DNA mismatch repair (MMR) is important for preventing mutations due to errors occurring during replication. Mismatches that are not removed by the proofreading function of the replicative DNA polymerases are recognized by a MutS protein in bacteria or usually in eukaryotes by a MutSα or MutSβ heterodimer.¹ After recognition, downstream events involving MutL in bacteria and usually MutLα in eukaryotes result in the removal of the newly synthesized DNA strand and resynthesis of the region, avoiding a change in DNA sequence.¹ However, there are many situations in which MMR is active outside of the context of genome replication, and an important question is what determines which strand of DNA is removed in those cases once a mismatch is detected. Using a reversion assay that detects point mutations in the TRP5 gene of yeast, we recently demonstrated that mispairs that escape detection at the replication fork can be recognized later by MMR and repaired in a manner independent of the replicating strand.² In that case, MMR was found to be acting in a mutagenic manner and could restore growth to cells that were in a nondividing state.When a mismatch is detected, how does MMR determine which strand to remove? A variety of experiments have demonstrated that eukaryotic MMR is directed to remove the DNA strand containing a nick, which could occur in the newly synthesized DNA strand through a variety of processes.^1,3 Evidence suggests that an important source of those nicks is activation of the latent endonuclease activity of MutLα; MutLα interacts with PCNA in a manner that could discriminate the template and primer stands of replication and thus give proper strand discrimination to MMR.⁴ Strand discrimination also likely involves MutSα and MutSβ, as both complexes have been shown to have robust interactions with PCNA.¹ However, two MMR pathways involving MutSα have recently been observed: one with MutSα as an integral component of replication factories and the other in which MutSα presumably scans the genome for mismatches and is independent of PCNA interaction.⁵ This latter pathway of MutSα, untethered to normal replication, is of interest here.One major function of MMR outside of replication is its anti-recombination role, by which it prevents aberrant recombination between non-identical sequences.⁶ MMR also functions in meiotic recombination and is responsible for the formation of gene conversion gradients, which can best be understood as arising from directed MMR near the site of initiating double-strand breaks, and randomly directed MMR further from the site of such strand discrimination signals.⁷ Although in meiotic recombination it appears that MMR acts without strand discrimination in some cases, the result is not an increase in mutation, as the end result is a choice between two pre-existing alleles.There are at least three situations in which MMR acting outside of replication appears to play a pro-mutagenic role. One is in the expansion of triplet repeat sequences, which play a role in a number of neurodegenerative diseases.^3,4 Although MMR would be expected to prevent repeat slippage during replication, there is biochemical evidence that slippage loops formed in triplet repeat sequences in a non-replicating state could load a PCNA-MutLα complex that would nick DNA in a random manner, leading to large expansions.⁴ There is also evidence from various mouse model systems that suggest a role for MMR in promoting repeat expansions.^3,8The second situation is somatic hypermutation. It had been found in 1998 that somatic hypermutation was, contrary to expectations, dependent on the presence of active MMR,⁹ but there was no mechanistic way to explain that dependence. A combination of patch replication by a relatively inaccurate translesion DNA polymerase and MMR acting randomly in terms of DNA strand, offers the best rationale for this process.^2,3The third situation, which also has the broadest sweep, is that of cancer. If several different pathways must be altered by mutation in order for a tumor to form, how could that happen in cells that either divide very slowly, or are in a nondividing state? Two years after the initial discovery of the linkage between MMR defects and cancer, MacPhee hypothesized that MMR acting in a “randomly templated” mode could be responsible for the formation of mutations in nondividing cells, which could reenter a growth phase as a result of those mutations;¹⁰ that corresponds to the observation we recently made in yeast, illustrated in Figure 1.² This is precisely the type of activity envisioned by MacPhee. What is not yet known is the signal giving strand discrimination to MMR; it could possibly be a random loading of MutLα,⁴ or it could be the presence of a random nick in the DNA strand close enough to the mismatch to be used for strand discrimination.Open in a separate windowFigure 1. Mutagenic MMR in nondividing cells. Cells were electroporated with 8-oxoGTP and then plated on Trp medium. The 8-oxoGTP was incorporated throughout the genome, including in some cells at the position indicated above which must revert via a TA→GC transversion in order for the cell to become Trp+. Once plated, the Trp- cells were unable to undergo even one round of replication, although they remained viable for over two weeks. In the absence of MMR, very few revertants appeared. However, in the presence of MMR, many revertants arose within the 3 d expected for cells that would have begun growth immediately after plating, but an even larger number of revertant colonies arose later on the plates up to a period of a week later. Thus only cells with active MMR were able to regain growth from a nondividing state due to the mutation created by MMR activity.The importance of MMR in preventing mutations during replication is unquestioned. However, the multiple activities of MMR outside of replication tend to be less appreciated. When MMR recognizes mismatches in DNA outside of the context of replication, the issue of what gives strand discrimination to MMR becomes critical. If the signal is randomly generated, MMR activity can be mutagenic, as we have found.²     

Mismatch repair of deaminated 5-methyl-cytosine

Deamination of 5-methyl-cytosine in double-stranded DNA produces a G-T mismatch. Heteroduplexes of bacteriophage lambda DNA containing a G-T mismatch at the site of a G-5-meC base-pair in one of the parental phages were constructed and used to transfect Escherichia coli cells. Genetic analysis of the progeny phages derived from such heteroduplexes suggests that, in E. coli, mismatches resulting from the deamination of 5-methyl-cytosine are repaired by a system requiring the E. coli dcm methylase and some, but not all, of the functions of the E. coli methyl-directed mismatch repair system. The repair appears to act only on the G-T mismatch and acts specifically to restore the cytosine methylation sequence.     

Mismatch repair activity in mammalian mitochondria

Mitochondrial DNA (mtDNA) defects cause debilitating metabolic disorders for which there is no effective treatment. Patients suffering from these diseases often harbour both a wild-type and a mutated subpopulation of mtDNA, a situation termed heteroplasmy. Understanding mtDNA repair mechanisms could facilitate the development of novel therapies to combat these diseases. In particular, mismatch repair activity could potentially be used to repair pathogenic mtDNA mutations existing in the heteroplasmic state if heteroduplexes could be generated. To date, however, there has been no compelling evidence for such a repair activity in mammalian mitochondria. We now report evidence consistent with a mismatch repair capability in mammalian mitochondria that exhibits some characteristics of the nuclear pathway. A repair assay utilising a nicked heteroduplex substrate with a GT or a GG mismatch in the β-galactosidase reporter gene was used to test the repair potential of different lysates. A low level repair activity was identified in rat liver mitochondrial lysate that showed no strand bias. The activity was mismatch-selective, bi-directional, ATP-dependent and EDTA-sensitive. Western analysis using antibody to MSH2, a key nuclear mismatch repair system (MMR) protein, showed no cross-reacting species in mitochondrial lysate. A hypothesis to explain the molecular mechanism of mitochondrial MMR in the light of these observations is discussed.     

Mismatch repair and DNA damage signalling

Postreplicative mismatch repair (MMR) increases the fidelity of DNA replication by up to three orders of magnitude, through correcting DNA polymerase errors that escaped proofreading. MMR also controls homologous recombination (HR) by aborting strand exchange between divergent DNA sequences. In recent years, MMR has also been implicated in the response of mammalian cells to DNA damaging agents. Thus, MMR-deficient cells were shown to be around 100-fold more resistant to killing by methylating agents of the S(N)1type than cells with functional MMR. In the case of cisplatin, the sensitivity difference was lower, typically two- to three-fold, but was observed in all matched MMR-proficient and -deficient cell pairs. More controversial is the role of MMR in cellular response to other DNA damaging agents, such as ionizing radiation (IR), topoisomerase poisons, antimetabolites, UV radiation and DNA intercalators. The MMR-dependent DNA damage signalling pathways activated by the above agents are also ill-defined. To date, signalling cascades involving the Ataxia telangiectasia mutated (ATM), ATM- and Rad3-related (ATR), as well as the stress-activated kinases JNK/SAPK and p38alpha have been linked with methylating agent and 6-thioguanine (TG) treatments, while cisplatin damage was reported to activate the c-Abl and JNK/SAPK kinases in MMR-dependent manner. MMR defects are found in several different cancer types, both familiar and sporadic, and it is possible that the involvement of the MMR system in DNA damage signalling play an important role in transformation. The scope of this article is to provide a brief overview of the recent literature on this subject and to raise questions that could be addressed in future studies.     

Mismatch repair proteins and mitotic genome stability

Mismatch repair (MMR) proteins play a critical role in maintaining the mitotic stability of eukaryotic genomes. MMR proteins repair errors made during DNA replication and in their absence, mutations accumulate at elevated rates. In addition, MMR proteins inhibit recombination between non-identical DNA sequences, and hence prevent genome rearrangements resulting from interactions between repetitive elements. This review provides an overview of the anti-mutator and anti-recombination functions of MMR proteins in the yeast Saccharomyces cerevisiae.     

Mismatch repair is diminished during stationary-phase mutation.

This paper is an invited Response to a recent Commentary [P.L. Foster, Rev. Mut. Res. 436 (1999) 179-184] entitled "Are adaptive mutations due to a decline in mismatch repair? The evidence is lacking". The Commentary argues that no evidence exists supporting the idea that mismatch repair is limiting specifically during stationary-phase mutation. A primary concern of the author is to question the method that we used previously to measure growth-dependent mutation. In this method, mutation rates are calculated using counts of mutant colonies taken at times when those colonies arise, rather than at a predetermined, fixed time. Here we show further data that illustrate why this must be done to ensure accurate mutation measurements. Such accuracy was necessary for our published determination that mismatch repair proteins are not limiting during growth-dependent mutation, but become so during stationary-phase mutation. We review the evidence supporting the idea that stationary-phase reversion of a lac frameshift mutation occurs in an environment of decreased mismatch repair capacity. Those data are substantial. The data presented in the Commentary, in apparent contradiction to this idea, do not justify the conclusion presented there.     

Mismatch repair in the antimutator Escherichia coli mud

Antimutators are genetic mutants that produce mutations at reduced rates compared to the wild type strain. They are interesting because they may provide insights into the mechanisms by which spontaneous mutations occur. We have investigated a reported antimutator strain of Escherichia coli termed mud for its possible mechanism. The mud strain exhibits a decrease in both spontaneous mutagenesis and mutability with alkylated agents and base analogs. These types of DNA lesions are known to be the substrates for the E. coli methyl-directed mismatch repair encoded by the mutHLSU system. We investigated whether the putative antimutator effect results from the increased expression or activity of the mutHLSU system. To directly measure the mismatch repair capacity of mud cells, we have transfected them with phage lambda heteroduplexes and scored the fraction of mixed (unrepaired) infective centers. This transfection system has been used routinely to assay mismatch repair capacity in E. coli and other organisms. No difference between mud and wild type cells is observed. From the results of the experiments we conclude that the reported antimutator effect of mud does not result from enhanced mismatch repair capacity. This conclusion is consistent with recently published evidence that the mud effect does not represent a real antimutator effect, but is an artifact due to impaired growth of mud cells under certain selective conditions.     

Mismatch repair: the praying hands of fidelity

High-resolution crystal structures have recently been solved for the mismatch binding protein MutS of Escherichia coli and its Thermus aquaticus homologue; they show how these factors recognise such structurally diverse substrates as base-base mismatches and insertion/deletion loops.     

Mismatch repair processing of carcinogen-DNA adducts triggers apoptosis

The DNA mismatch repair pathway is well known for its role in correcting biosynthetic errors of DNA replication. We report here a novel role for mismatch repair in signaling programmed cell death in response to DNA damage induced by chemical carcinogens. Cells proficient in mismatch repair were highly sensitive to the cytotoxic effects of chemical carcinogens, while cells defective in either human MutS or MutL homologs were relatively insensitive. Since wild-type cells but not mutant cells underwent apoptosis upon treatment with chemical carcinogens, the apoptotic response is dependent on a functional mismatch repair system. By analyzing p53 expression in several pairs of cell lines, we found that the mismatch repair-dependent apoptotic response was mediated through both p53-dependent and p53-independent pathways. In vitro biochemical studies demonstrated that the human mismatch recognition proteins hMutSalpha and hMutSbeta efficiently recognized DNA damage induced by chemical carcinogens, suggesting a direct participation of mismatch repair proteins in mediating the apoptotic response. Taken together, these studies further elucidate the mechanism by which mismatch repair deficiency predisposes to cancer, i.e., the deficiency not only causes a failure to repair mismatches generated during DNA metabolism but also fails to direct damaged and mutation-prone cells to commit suicide.