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

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

Specific repair systems are activated in response to the DNA damage. Mismatch repair protects the genome of prokaryotic and eukaryotic cells from lesions that appear during process of DNA replication or are induced by mutagenic factors. The methyl directed mismatch repair distinguishes the new strand from the old strand by the hemi-methylated state of the DNA and controls the fidelity of genetic information after homologous recombination. The very short patch repair restores the mismatches at the sites with nucleotide sequence CC(W/T)GG. The "8-oxoG" pathway is independent of the hemi-methylated state of the DNA, and removes the oxidated nucleotides from the genome of prokaryotes and eukaryotes. Mutations in genes of mismatch repair enhance the process of mutagenesis in prokaryotic cell, and are the reason for the development of the colon cancer in humans. The mechanisms of mismatch repair and the role of defective repair proteins in mutagenesis and carcinogenesis are discussed in this review.     

DNA repair defects in colon cancer

Defects in DNA-repair pathways lead to an accumulation of mutations in genomic DNA that result from non-repair or mis-repair of modifications introduced into the DNA by endogenous or exogenous agents or by the malfunction of DNA metabolic pathways. Until recently, only two repair pathways, postreplicative mismatch repair and nucleotide excision repair, have been linked to cancer in mammals, but these have been joined in recent months also by the damage-reversal and base-excision-repair processes, which have been shown to be inactivated, either through mutation or epigenetically, in human cancer.     

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 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 outside of replication

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

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 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 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 protein MSH2 expression and prognosis of colorectal cancer patients

INTRODUCTION AND AIMS: The role of genetic factors in the etiology and prognosis of patients with sporadic colorectal cancer is controversial. We have therefore investigated the biological and clinicopathological influence of immunohistochemical MSH2 expression in colorectal cancer. PATIENTS AND METHODS: A total of 49 consecutive patients with unselected colorectal cancer operated on in our unit were included in the study. All tumors were resected and tumor specimens were evaluated for MSH2 expression. Clinicopathological data and patient survival were correlated with MSH2 staining. Uni- and multivariate analyses were performed. The minimum follow-up period was five years. RESULTS: Curative resection was performed in 34 patients (64.9%), 14 of whom subsequently relapsed. At the end of the overall follow-up 25 (51%) patients had died, 21 of cancer-related causes. Twenty-eight patients (57.1%) were negative for MSH2 staining. Only vascular invasion was significantly correlated with MSH2 expression (lower median values; p=0.04). The overall median survival was 47.9 months (95% CI=27-86.6%). Multivariate analysis of variables in relation to survival showed that T stage (p=0.001), N stage (p<0.001) and MSH2 expression (p=0.01) were independent factors for survival. CONCLUSIONS: Reduced MSH2 expression is frequent in unselected colorectal cancer patients. Only vascular invasion was correlated with MSH2 expression in this study. Survival was related to TN stage and MSH2 staining.     

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 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.     

Approaches to diagnose DNA mismatch repair gene defects in cancer

The DNA repair pathway mismatch repair (MMR) is responsible for the recognition and correction of DNA biosynthetic errors caused by inaccurate nucleotide incorporation during replication. Faulty MMR leads to failure to address the mispairs or insertion deletion loops (IDLs) left behind by the replicative polymerases and results in increased mutation load at the genome. The realization that defective MMR leads to a hypermutation phenotype and increased risk of tumorigenesis highlights the relevance of this pathway for human disease. The association of MMR defects with increased risk of cancer development was first observed in colorectal cancer patients that carried inactivating germline mutations in MMR genes and the disease was named as hereditary non-polyposis colorectal cancer (HNPCC). Currently, a growing list of cancers is found to be MMR defective and HNPCC has been renamed Lynch syndrome (LS) partly to include the associated risk of developing extra-colonic cancers. In addition, a number of non-hereditary, mostly epigenetic, alterations of MMR genes have been described in sporadic tumors. Besides conferring a strong cancer predisposition, genetic or epigenetic inactivation of MMR genes also renders cells resistant to some chemotherapeutic agents. Therefore, diagnosis of MMR deficiency has important implications for the management of the patients, the surveillance of their relatives in the case of LS and for the choice of treatment. Some of the alterations found in MMR genes have already been well defined and their pathogenicity assessed. Despite this substantial wealth of knowledge, the effects of a large number of alterations remain uncharacterized (variants of uncertain significance, VUSs). The advent of personalized genomics is likely to increase the list of VUSs found in MMR genes and anticipates the need of diagnostic tools for rapid assessment of their pathogenicity. This review describes current tools and future strategies for addressing the relevance of MMR gene alterations in human disease.     

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 genes and microsatellite instability as molecular markers for gynecological cancer detection

Due to major developments in genetics over the past decade, molecular biology tests are serving promising tools in early diagnosis and follow-up of cancer patients. Recent epidemiological studies revealed that the risk for each individual to develop cancer is closely linked to his/her own genetic potentialities. Some populations that are defective in DNA repair processes, for example in Xeroderma pigmentosum or in the Lynch syndrome, are particularly prone to cancer due to the accumulation of mutations within the genome. Such populations would benefit from the development of tests aimed at identifying people who are particularly at risk. Here, we review some data suggesting that the inactivation of mismatch repair is often found in endometrial cancer and we discuss molecular-based strategies that would help to identify the affected individuals in families with cases of glandular malignancies.     

Mismatch repair and recombination in E. coli

The involvement of the E. coli methyl-directed and very short patch (vsp) mismatch repair systems in bacteriophage lambda recombination has been studied. Genetic crosses and heteroduplex transfection experiments were performed using lambda phages with sequenced mutations in the cl gene. The results indicate that methyl-directed repair does operate during bacteriophage lambda recombination but generally does not contribute to the formation of recombinants involving close markers. Vsp repair apparently acts during bacteriophage lambda recombination to produce recombinants involving close markers because its action does not involve extensive excision tracts. Marker-specific hyperrecombination and the apparent clustering of genetic exchanges in bacteriophage lambda recombination can be accounted for by the action of the vsp repair system.