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 outside of replication

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

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 participates in error-free processing of DNA interstrand crosslinks in human cells

DNA interstrand crosslinks (ICLs) present formidable blocks to DNA metabolic processes and must be repaired for cell survival. ICLs are induced in DNA by intercalating compounds such as the widely used therapeutic agent psoralen. In bacteria, both nucleotide excision repair (NER) and homologous recombination are required for the repair of ICLs. The processing of ICLs in mammalian cells is not clearly understood. However, it is known that processing can occur by NER, which for psoralen ICLs can be an error-generating process conducive to mutagenesis. We show here that another repair pathway, mismatch repair (MMR), is also involved in eliminating psoralen ICLs in human cells. MMR deficiency renders cells hypersensitive to psoralen ICLs without diminishing their mutagenic potential, suggesting that MMR does not contribute to error-generating repair, and that MMR may represent a relatively error-free mechanism for processing these lesions in human cells. Thus, enhancement of MMR relative to NER may reduce the mutagenesis caused by DNA ICLs in humans.     

Evidence for preferential mismatch repair of lagging strand DNA replication errors in yeast

Duplex DNA is replicated in the 5'-3' direction by coordinated copying of leading and lagging strand templates with somewhat different proteins and mechanics, providing the potential for differences in the fidelity of replication of the two strands. We previously showed that in Saccharomyces cerevisiae, active replication origins establish a strand bias in the rate of base substitutions resulting from replication of unrepaired 8-oxo-guanine (GO) in DNA. Lower mutagenesis was associated with replicating lagging strand templates. Here, we test the hypothesis that this bias is due to more efficient repair of lagging stand mismatches by measuring mutation rates in ogg1 strains with a reporter allele in two orientations at loci on opposite sides of a replication origin on chromosome III. We compare a MMR-proficient strain to strains deleted for the MMR genes MSH2, MSH6, MLH1, or EXOI. Loss of MMR reduces the strand bias by preferentially increasing mutagenesis for lagging strand replication. We conclude that GO-A mismatches generated during lagging strand replication are more efficiently repaired. This is consistent with the hypothesis that 5' ends of Okazaki fragments and PCNA, present at high density during lagging strand replication, are used as strand discrimination signals for mismatch repair in vivo.     

Reactive oxygen-mediated damage to a human DNA replication and repair protein

Ultraviolet A (UVA) makes up more than 90% of incident terrestrial ultraviolet radiation. Unlike shorter wavelength UVB, which damages DNA directly, UVA is absorbed poorly by DNA and is therefore considered to be less hazardous. Organ transplant patients treated with the immunosuppressant azathioprine frequently develop skin cancer. Their DNA contains 6-thioguanine-a base analogue that generates DNA-damaging singlet oxygen ((1)O(2)) when exposed to UVA. Here, we show that this (1)O(2) damages proliferating cell nuclear antigen (PCNA), the homotrimeric DNA polymerase sliding clamp. It causes covalent oxidative crosslinking between the PCNA subunits through a histidine residue in the intersubunit domain. Crosslinking also occurs after treatment with higher-although still moderate-doses of UVA alone or with chemical oxidants. Chronic accumulation of oxidized proteins is linked to neurodegenerative disorders and ageing. Our findings identify oxidative damage to an important DNA replication and repair protein as a previously unrecognized hazard of acute oxidative stress.     

Mismatch repair causes the dynamic release of an essential DNA polymerase from the replication fork

Mismatch repair (MMR) corrects DNA polymerase errors occurring during genome replication. MMR is critical for genome maintenance, and its loss increases mutation rates several hundred fold. Recent work has shown that the interaction between the mismatch recognition protein MutS and the replication processivity clamp is important for MMR in Bacillus subtilis. To further understand how MMR is coupled to DNA replication, we examined the subcellular localization of MMR and DNA replication proteins fused to green fluorescent protein (GFP) in live cells, following an increase in DNA replication errors. We demonstrate that foci of the essential DNA polymerase DnaE-GFP decrease following mismatch incorporation and that loss of DnaE-GFP foci requires MutS. Furthermore, we show that MutS and MutL bind DnaE in vitro, suggesting that DnaE is coupled to repair. We also found that DnaE-GFP foci decrease in vivo following a DNA damage-independent arrest of DNA synthesis showing that loss of DnaE-GFP foci is caused by perturbations to DNA replication. We propose that MutS directly contacts the DNA replication machinery, causing a dynamic change in the organization of DnaE at the replication fork during MMR. Our results establish a striking and intimate connection between MMR and the replicating DNA polymerase complex in vivo.     

A SUMOry of DNA replication: synthesis, damage, and repair

Recombination at stalled replication forks is regulated at an early stage by sumoylation. In this issue of Cell, Branzei et al. show that the Ubc9/SUMO modification pathway controls the accumulation of cruciform structures at stalled forks.     

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.     

Absence of DNA repair replication after chemical mutagen damage in Vicia faba

Exposure of human (Hela) cells to the mutagens 4-nitroquinoline-1-oxide (4NQO) and N-methyl-N′-nitro-nitrosoguanidine (MNNG) produces damage in DNA that is repaired by a mechanism involving the insertion of new bases into DNA (repair replication). Vicia faba root tips, either from soaked seeds containing non-proliferating cells or from growing roots, do not perform detectable amounts of repair replication even though the mutagens inhibit DNA synthesis and cause chromosome aberrations. In view of similar failures to resolve excision in Chlamydomonas, Haplopappus, and Nicotiana after irradiation with UV light and in Vicia faba after X-irradiation it appears that plants in general might lack this repair process.     

Eukaryotic DNA polymerases in DNA replication and DNA repair

DNA polymerases carry out a large variety of synthetic transactions during DNA replication, DNA recombination and DNA repair. Substrates for DNA polymerases vary from single nucleotide gaps to kilobase size gaps and from relatively simple gapped structures to complex replication forks in which two strands need to be replicated simultaneously. Consequently, one would expect the cell to have developed a well-defined set of DNA polymerases with each one uniquely adapted for a specific pathway. And to some degree this turns out to be the case. However, in addition we seem to find a large degree of cross-functionality of DNA polymerases in these different pathways. DNA polymerase α is almost exclusively required for the initiation of DNA replication and the priming of Okazaki fragments during elongation. In most organisms no specific repair role beyond that of checkpoint control has been assigned to this enzyme. DNA polymerase δ functions as a dimer and, therefore, may be responsible for both leading and lagging strand DNA replication. In addition, this enzyme is required for mismatch repair and, together with DNA polymerase ζ, for mutagenesis. The function of DNA polymerase ɛ in DNA replication may be restricted to that of Okazaki fragment maturation. In contrast, either polymerase δ or ɛ suffices for the repair of UV-induced damage. The role of DNA polymerase β in base-excision repair is well established for mammalian systems, but in yeast, DNA polymerase δ appears to fullfill that function. Received: 20 April 1998 / Accepted: 8 May 1998     

Archaeal DNA replication and repair

Since the first archaeal genome was sequenced, much attention has been focused on the study of these unique microorganisms. We have learnt that although archaeal DNA metabolic processes (replication, recombination and repair) are more similar to the metabolic processes of Eukarya than those of Bacteria, Archaea are not simply 'mini Eukarya'. They are, in fact, a mosaic of the eukaryal and bacterial systems that also possess archaeal-specific features. Recent biochemical and structural studies of the proteins that participate in archaeal DNA replication and repair have increased our understanding of these processes.     

DNA ligases in the repair and replication of DNA

DNA ligases are critical enzymes of DNA metabolism. The reaction they catalyse (the joining of nicked DNA) is required in DNA replication and in DNA repair pathways that require the re-synthesis of DNA.Most organisms express DNA ligases powered by ATP, but eubacteria appear to be unique in having ligases driven by NAD(+). Interestingly, despite protein sequence and biochemical differences between the two classes of ligase, the structure of the adenylation domain is remarkably similar. Higher organisms express a variety of different ligases, which appear to be targetted to specific functions. DNA ligase I is required for Okazaki fragment joining and some repair pathways; DNA ligase II appears to be a degradation product of ligase III; DNA ligase III has several isoforms, which are involved in repair and recombination and DNA ligase IV is necessary for V(D)J recombination and non-homologous end-joining. Sequence and structural analysis of DNA ligases has shown that these enzymes are built around a common catalytic core, which is likely to be similar in three-dimensional structure to that of T7-bacteriophage ligase. The differences between the various ligases are likely to be mediated by regions outside of this common core, the structures of which are not known. Therefore, the determination of these structures, along with the structures of ligases bound to substrate DNAs and partner proteins ought to be seen as a priority.