DNA mismatch repair and mutation avoidance pathways

Unpaired and mispaired bases in DNA can arise by replication errors, spontaneous or induced base modifications, and during recombination. The major pathway for correction of mismatches arising during replication is the MutHLS pathway of Escherichia coli and related pathways in other organisms. MutS initiates repair by binding to the mismatch, and activates together with MutL the MutH endonuclease, which incises at hemimethylated dam sites and thereby mediates strand discrimination. Multiple MutS and MutL homologues exist in eukaryotes, which play different roles in the mismatch repair (MMR) pathway or in recombination. No MutH homologues have been identified in eukaryotes, suggesting that strand discrimination is different to E. coli. Repair can be initiated by the heterodimers MSH2-MSH6 (MutSalpha) and MSH2-MSH3 (MutSbeta). Interestingly, MSH3 (and thus MutSbeta) is missing in some genomes, as for example in Drosophila, or is present as in Schizosaccharomyces pombe but appears to play no role in MMR. MLH1-PMS1 (MutLalpha) is the major MutL homologous heterodimer. Again some, but not all, eukaryotes have additional MutL homologues, which all form a heterodimer with MLH1 and which play a minor role in MMR. Additional factors with a possible function in eukaryotic MMR are PCNA, EXO1, and the DNA polymerases delta and epsilon. MMR-independent pathways or factors that can process some types of mismatches in DNA are nucleotide-excision repair (NER), some base excision repair (BER) glycosylases, and the flap endonuclease FEN-1. A pathway has been identified in Saccharomyces cerevisiae and human that corrects loops with about 16 to several hundreds of unpaired nucleotides. Such large loops cannot be processed by MMR.     

DNA mismatch repair system. Classical and fresh roles

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 framework 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, mutations 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.     

A Titin mutation defines roles for circulation in endothelial morphogenesis

Morphogenesis of the developing vascular network requires coordinated regulation of an extensive array of endothelial cell behaviors. Precisely regulated signaling molecules such as vascular endothelial growth factor (VEGF) direct some of these endothelial behaviors. Newly forming blood vessels also become subjected to novel biomechanical forces upon initiation of cardiac contractions. We report here the identification of a recessive mouse mutation termed shrunken-head (shru) that disrupts function of the Titin gene. Titin was found to be required for the initiation of proper heart contractions as well as for maintaining the correct overall shape and orientation of individual cardiomyocytes. Cardiac dysfunction in shrunken-head mutant embryos provided an opportunity to study the effects of lack of blood circulation on the morphogenesis of endothelial cells. Without blood flow, differentiating endothelial cells display defects in their shapes and patterns of cell-cell contact. These endothelial cells, without exposure to blood circulation, have an abnormal distribution within vasculogenic vessels. Further effects of absent blood flow include abnormal spatial regulation of angiogenesis and elevated VEGF signaling. The shrunken-head mutation has provided an in vivo model to precisely define the roles of circulation on cellular and network aspects of vascular morphogenesis.     

Strand targeting signal(s) for in vivo mutation avoidance by post-replication mismatch repair in Escherichia coli

Summary The involvement of GATC sites in directing mismatch correction for the elimination of replication errors in Escherichia coli was investigated in vivo by analyzing mutation rates for a gene carried on a series of related plasmids that contain 2, 1 and 0 such sites. This gene encoding chloramphenicol acetyl transferase (Cat protein) was inactivated by a point mutation. In vivo mutations restoring resistance to chloramphenicol were scored in mismatch repair proficient (mut ⁺) and deficient (mutHLS^-) strains. In mut ⁺ cells, reduction of GATC sites from 2 to 0 increased mutation rates approximately 10-fold. Removal of the GATC site distal to the cat ^- mutation increased the rate of mutation less than 2-fold, indicating that mismatch repair can proceed normally with a single site. The mutation rate increased 3-fold after removal of the GATC site proximal to the mutation. In the absence of a GATC site, mutL^- and mutS^- strains exhibited a 2- to 3-fold increased mutation rate as compared to isogenic mutH^- and mut ⁺ strains. This indicates that 50%–70% of replication errors can be corrected in a mutLS-dependent way in the absence of any GATC site to target mismatch correction to newly synthesized DNA strands. Other strand targeting signals, possibly single strand discontinuities, might be used in mutLS-dependent repair     

DNA mismatch repair and cancer

Five human DNA mismatch repair genes have been identified that, when mutated, cause susceptibility to hereditary nonpolyposis colorectal cancer (HNPCC). Mutational inactivation of both copies of a DNA mismatch repair gene results in a profound repair defect and progressive accumulation of mutations throughout the genome. Some of the mutations confer selective advantage on the cells, giving rise to cancer. Recent discoveries suggest that apart from postreplication repair, DNA mismatch repair proteins have several other functions that are highly relevant to carcinogenesis. These include DNA damage surveillance, prevention of recombination between nonidentical sequences and participation in meiotic processes (chromosome pairing). A brief overview of these different features of the human DNA mismatch repair system will be provided, with the emphasis in their implications in cancer development.     

Eukaryotic DNA mismatch repair

Eukaryotic mismatch repair (MMR) has been shown to require two different heterodimeric complexes of MutS-related proteins: MSH2-MSH3 and MSH2-MSH6. These two complexes have different mispair recognition properties and different abilities to support MMR. Alternative models have been proposed for how these MSH complexes function in MMR. Two different heterodimeric complexes of MutL-related proteins, MLH1-PMS1 (human PMS2) and MLH1-MLH3 (human PMS1) also function in MMR and appear to interact with other MMR proteins including the MSH complexes and replication factors. A number of other proteins have been implicated in MMR, including DNA polymerase delta, RPA (replication protein A), PCNA (proliferating cell nuclear antigen), RFC (replication factor C), Exonuclease 1, FEN1 (RAD27) and the DNA polymerase delta and epsilon associated exonucleases. MMR proteins have also been shown to function in other types of repair and recombination that appear distinct from MMR. MMR proteins function in these processes in conjunction with components of nucleotide excision repair (NER) and, possibly, recombination.     

Frameshift mutation, microsatellites and mismatch repair

The structure of eukaryotic DNA, with its repeated sequences, makes base addition and loss a major obstacle to the maintenance of genetic stability. As compared to the bacteria, much of the mismatch repair capacity of the eukaryotic cell must be devoted to the surveillance of frameshift changes. Any alteration in the activity of proteins which recognize frameshifts or which hold the DNA in place during replication is likely to result in genomic instability.     

Exonuclease 1 and its versatile roles in DNA repair

Exonuclease 1 (EXO1) is a multifunctional 5′ → 3′ exonuclease and a DNA structure-specific DNA endonuclease. EXO1 plays roles in DNA replication, DNA mismatch repair (MMR) and DNA double-stranded break repair (DSBR) in lower and higher eukaryotes and contributes to meiosis, immunoglobulin maturation, and micro-mediated end-joining in higher eukaryotes. In human cells, EXO1 is also thought to play a role in telomere maintenance. Mutations in the human EXO1 gene correlate with increased susceptibility to some cancers. This review summarizes recent studies on the enzymatic functions and biological roles of EXO1, its possible protective role against cancer and aging, and regulation of EXO1 by posttranslational modification.     

DNA mismatch repair and Lynch syndrome

The evolutionary conserved mismatch repair proteins correct a wide range of DNA replication errors. Their importance as guardians of genetic integrity is reflected by the tremendous decrease of replication fidelity (two to three orders of magnitude) conferred by their loss. Germline mutations in mismatch repair genes, predominantly MSH2 and MLH1, have been found to underlie the Lynch syndrome (also called hereditary non-polyposis colorectal cancer, HNPCC), a hereditary predisposition for cancer. Lynch syndrome affects predominantly the colon and accounts for 2–5% of all colon cancer cases. During more than 30 years of biochemical, crystallographic and clinical research, deep insight has been achieved in the function of mismatch repair and the diseases that are associated with its loss. We review the biochemistry of mismatch repair and also introduce the clinical, diagnostic and genetic aspects of Lynch syndrome.     

DNA binding by yeast Mlh1 and Pms1: implications for DNA mismatch repair

The yeast Mlh1–Pms1 heterodimer required for mismatch repair (MMR) binds to DNA. Here we map DNA binding to N-terminal fragments of Mlh1 and Pms1. We demonstrate that Mlh1 and Pms1 N-terminal domains (NTDs) independently bind to double-stranded and single-stranded DNA, in the absence of dimerization and with different affinities. Full-length Mlh1p alone, which can homodimerize, also binds to DNA. Substituting conserved positively charged amino acids in Mlh1 produces mutator phenotypes in a haploid yeast strain characteristic of reduced MMR. These substitutions strongly reduce DNA binding by the Mlh1 NTD and, to a lesser extent, they also reduce DNA binding by full-length Mlh1 and the Mlh1–Pms1 heterodimer. Replacement of a homologous Pms1 residue has a much smaller effect on mutation rate and does not reduce DNA binding. The results demonstrate that NTDs of yeast Mlh1 and Pms1 contain independent DNA binding sites and they suggest that the C-terminal region of Mlh1p may also contribute to DNA binding. The differential mutator effects and binding properties observed here further suggest that Mlh1 and Pms1 differ in their interactions with DNA. Finally, the results are consistent with the hypothesis that DNA binding by Mlh1 is important for MMR.     

EXO1 and MSH6 are high-copy suppressors of conditional mutations in the MSH2 mismatch repair gene of Saccharomyces cerevisiae

In Saccharomyces cerevisiae, Msh2p, a central component in mismatch repair, forms a heterodimer with Msh3p to repair small insertion/deletion mismatches and with Msh6p to repair base pair mismatches and single-nucleotide insertion/deletion mismatches. In haploids, a msh2Delta mutation is synthetically lethal with pol3-01, a mutation in the Poldelta proofreading exonuclease. Six conditional alleles of msh2 were identified as those that conferred viability in pol3-01 strains at 26 degrees but not at 35 degrees. DNA sequencing revealed that mutations in several of the msh2(ts) alleles are located in regions with previously unidentified functions. The conditional inviability of two mutants, msh2-L560S pol3-01 and msh2-L910P pol3-01, was suppressed by overexpression of EXO1 and MSH6, respectively. Partial suppression was also observed for the temperature-sensitive mutator phenotype exhibited by msh2-L560S and msh2-L910P strains in the lys2-Bgl reversion assay. High-copy plasmids bearing mutations in the conserved EXO1 nuclease domain were unable to suppress msh2-L560S pol3-01 conditional lethality. These results, in combination with a genetic analysis of msh6Delta pol3-01 and msh3Delta pol3-01 strains, suggest that the activity of the Msh2p-Msh6p heterodimer is important for viability in the presence of the pol3-01 mutation and that Exo1p plays a catalytic role in Msh2p-mediated mismatch repair.     

Methyl-directed DNA mismatch repair in Escherichia coli

Some of the molecular aspects of methyl-directed mismatch repair in E. coli have been characterized. These include: mismatch recognition by mutS protein in which different mispairs are bound with different affinities; the direct involvement of d(GATC) sites; and strand scission by mutH protein at d(GATC) sequences with strand selection based on methylation of the DNA at those sites. In addition, communication over a distance between a mismatch and d(GATC) sites has been implicated. Analysis of mismatch correction in a defined system (Lahue et al., unpublished) should provide a direct means to further molecular aspects of this process.     

DNA mismatch repair in trinucleotide repeat instability

Trinucleotide repeat expansions cause over 30 severe neuromuscular and neurodegenerative disorders, including Huntington's disease, myotonic dystrophy type 1, and fragile X syndrome. Although previous studies have substantially advanced the understanding of the disease biology, many key features remain unknown. DNA mismatch repair(MMR) plays a critical role in genome maintenance by removing DNA mismatches generated during DNA replication. However, MMR components,particularly mismatch recognition protein MutSβ and its interacting factors MutLα and MutLγ, have been implicated in trinucleotide repeat instability. In this review, we will discuss the roles of these key MMR proteins in promoting trinucleotide repeat instability.     

Exo1 independent DNA mismatch repair involves multiple compensatory nucleases

Functional DNA mismatch repair (MMR) is essential for maintaining the fidelity of DNA replication and genetic stability. In hematopoiesis, loss of MMR results in methylating agent resistance and a hematopoietic stem cell (HSC) repopulation defect. Additionally MMR failure is associated with a variety of human malignancies, notably Lynch syndrome. We focus on the 5′ → 3′ exonuclease Exo1, the primary enzyme excising the nicked strand during MMR, preceding polymerase synthesis. We found that nuclease dead Exo1 mutant cells are sensitive to the O6-methylguanine alkylating agent temozolomide when given with the MGMT inactivator, O6benzylguanine (BG). Additionally we used an MMR reporter plasmid to verify that Exo1^mut MEFs were able to repair G:T base mismatches in vitro. We showed that unlike other MMR deficient mouse models, Exo1^mut mouse HSC did not gain a competitive survival advantage post temozolomide/BG treatment in vivo. To determine potential nucleases implicated in MMR in the absence of Exo1 nuclease activity, but in the presence of the inactive protein, we performed gene expression analyses of several mammalian nucleases in WT and Exo1^mut MEFs before and after temozolomide treatment and identified upregulation of Artemis, Fan1, and Mre11. Partial shRNA mediated silencing of each of these in Exo1^mut cells resulted in decreased MMR capacity and increased resistance to temozolomide/BG. We propose that nuclease function is required for fully functional MMR, but a portfolio of nucleases is able to compensate for loss of Exo1 nuclease activity to maintain proficiency.     

Mechanisms and functions of DNA mismatch repair

DNA mismatch repair （MMR） is a highly conserved biological pathway that plays a key role in maintaining genomic stability. The specificity of MMR is primarily for base-base mismatches and insertion/deletion mispairs generated during DNA replication and recombination. MMR also suppresses homeologous recombination and was recently shown to play a role in DNA damage signaling in eukaryotic cells. Escherichia coli MutS and MutL and their eukaryotic homologs, MutSα and MutLα, respectively, are key players in MMR-associated genome maintenance. Many other protein components that participate in various DNA metabolic pathways, such as PCNA and RPA, are also essential for MMR. Defects in MMR are associated with genome-wide instability, predisposition to certain types of cancer including hereditary non-polyposis colorectal cancer, resistance to certain chemotherapeutic agents, and abnormalities in meiosis and sterility in mammalian systems.     

The cell cycle and DNA mismatch repair

The DNA mismatch repair (MMR) pathway contributes to the fidelity of DNA synthesis and recombination by correcting mispaired nucleotides and insertion/deletion loops (IDLs). We have investigated whether MMR protein expression, activity, and subcellular location are altered during discrete phases of the cell cycle in mammalian cells. Two distinct methods have been used to demonstrate that although physiological MMR protein expression, mismatch binding, and nick-directed MMR activity within the nucleus are at highest levels during S phase, MMR is active throughout the cell cycle. Despite equal MMR nuclear protein concentrations in S and G(2) phases, mismatch binding and repair activities within G(2) are significantly lower, indicating a post-translational decrease in MMR activity specific to G(2). We further demonstrate that typical co-localization of MutSalpha to late S phase replication foci can be disrupted by 2 microM N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). This concentration of MNNG does not decrease ongoing DNA synthesis nor induce cell cycle arrest until the second cell cycle, with long-term colony survival decreased by only 24%. These results suggest that low level alkylation damage can selectively disrupt MMR proofreading activity during DNA synthesis and potentially increase mutation frequency within surviving cells.     

Synergistic effect of TRM2/RNC1 and EXO1 in DNA double-strand break repair in Saccharomyces cerevisiae

In our recently published study, we provided in vitro as well as in vivo data demonstrating the involvement of TRM2/RNC1 in homologous recombination based repair (HRR) of DNA double strand breaks (DSBs), in support of such claims reported earlier. To further validate its role in DNA DSB processing, our present study revealed that the trm2 single mutant displays higher sensitivity to persistent induction of specific DSBs at the MAT locus by HO-endonuclease with higher sterility rate among the survivors compared to wild type (wt) or exo1 single mutants. Intriguingly, both sensitivity and sterility rate increased dramatically in trm2exo1 double mutants lacking both endo-exonucleases with a progressively increased sterility rate in trm2exo1 double mutants with short-induction periods, reaching a very high level of sterility with persistent DSB inductions. Mutation analysis of the mating type (MAT) locus among the sterile survivors with persistent HO-induction in trm2 and exo1 single mutants as well as in trm2exo1 double mutants revealed a similar small insertions and deletions events, characteristic of non-homologous end joining (NHEJ) that might have occurred due to the lack of proper processing function in these mutants. In addition, trm2ku80 and trm2rad52 double mutants also displayed significantly higher sterility with persistent DSB induction compared to ku80 and rad52 single mutants, respectively, exhibiting a mutation spectra that shifted from base substitution (in ku80 and rad52 single mutants) to small insertions and deletions in the double mutants (in trm2ku80 and trm2rad52 mutants). These data indicate a defective processing in absence of TRM2, with a synergistic effect of TRM2, and EXO1 in such processing.     

Distinct roles for the Saccharomyces cerevisiae mismatch repair proteins in heteroduplex rejection, mismatch repair and nonhomologous tail removal

The Saccharomyces cerevisiae mismatch repair (MMR) protein MSH6 and the SGS1 helicase were recently shown to play similarly important roles in preventing recombination between divergent DNA sequences in a single-strand annealing (SSA) assay. In contrast, MMR factors such as Mlh1p, Pms1p, and Exo1p were shown to not be required or to play only minimal roles. In this study we tested mutations that disrupt Sgs1p helicase activity, Msh2p-Msh6p mismatch recognition, and ATP binding and hydrolysis activities for their effect on preventing recombination between divergent DNA sequences (heteroduplex rejection) during SSA. The results support a model in which the Msh proteins act with Sgs1p to unwind DNA recombination intermediates containing mismatches. Importantly, msh2 mutants that displayed separation-of-function phenotypes with respect to nonhomologous tail removal during SSA and heteroduplex rejection were characterized. These studies suggest that nonhomologous tail removal is a separate function of Msh proteins that is likely to involve a distinct DNA binding activity. The involvement of Sgs1p in heteroduplex rejection but not nonhomologous tail removal further illustrates that subsets of MMR proteins collaborate with factors in different DNA repair pathways to maintain genome stability.