The mismatch repair (MMR) system detects non-Watson-Crick base pairs and strand misalignments arising during DNA replication and mediates their removal by catalyzing excision of the mispair-containing tract of nascent DNA and its error-free resynthesis. In this way, MMR improves the fidelity of replication by several orders of magnitude. It also addresses mispairs and strand misalignments arising during recombination and prevents synapses between nonidentical DNA sequences. Unsurprisingly, MMR malfunction brings about genomic instability that leads to cancer in mammals. But MMR proteins have recently been implicated also in other processes of DNA metabolism, such as DNA damage signaling, antibody diversification, and repair of interstrand cross-links and oxidative DNA damage, in which their functions remain to be elucidated. This article reviews the progress in our understanding of the mechanism of replication error repair made during the past decade.

AbstractDNA methylation at C of CpG dyads (mCpG) in vertebrate genomes is essential for gene regulation, genome stability and development. We show in this study that proper functioning of post-replicative DNA mismatch repair (MMR) in mammalian cells relies on the presence of genomic mCpG, as well as on the maintenance DNA methyltransferase Dnmt1 independently of its catalytic activity. More importantly, high efficiency of mammalian MMR surveillance is achieved through a hemi-mCpG-Np95(Uhrf1)-Dnmt1 axis, in which the MMR surveillance complex(es) is recruited to post-replicative DNA by Dnmt1, requiring its interactions with MutSα, as well as with Np95 bound at the hemi-methylated CpG sites. Thus, efficiency of MMR surveillance over the mammalian genome in vivo is enhanced at the epigenetic level. This synergy endows vertebrate CpG methylation with a new biological significance and, consequently, an additional mechanism for the maintenance of vertebrate genome stability.

Endogenous barriers to DNA replication, such as repetitive DNA, non-B DNA structures, and protein barriers present significant challenges to replication. Upon encountering one of these barriers, cells employ a number of strategies to ensure completion of replication. Some of these pathways operate at the stalled replication fork and others occur post-replicatively. These pathways vary both in their timing and the nuclear location in which they occur. Here we review how cells deal with endogenous sources of replication stress, with a focus on structure-forming DNA repeats, and our current understanding of how cells use nuclear positioning to facilitate the repair of natural replication barriers.

DNA secondary structures, such as hairpins, cruciforms, triplexes, G-quadruplexes and iMotifs, are common, dynamic features that replication forks routinely encounter. However, how these structures destabilise the replication fork remains unclear. Here, we propose a framework describing the immediate consequences of replication forks encountering DNA secondary structures. This review considers outcomes according to the affected strand (leading or lagging) and the timing of structure formation, linking strand geometry and folding dynamics to replisome behaviour. Stable, pre-formed structures on the leading strand template either impede, or are bypassed by, the CMG (CDC45-MCM-GINS) helicase, frequently leaving single-stranded DNA (ssDNA) gaps. Leading strand structures inhibit DNA polymerase ε (Pol ε), induce fork uncoupling, again producing post-replicative ssDNA gaps which can channel into fork reversal or PrimPol-dependent repriming. Lagging strand template structures inhibit DNA polymerase δ (Pol δ) and structures on 5' flaps impair Okazaki fragment maturation (OFM); both impediments yield ssDNA nicks or gaps. In each case, replication protein A (RPA) availability and the replication checkpoint define a tolerance window and coordinate hand-offs to accessory helicases, Pol δ strand displacement synthesis, and translesion synthesis (TLS). Immediate double-strand breaks (DSBs) are unlikely as an immediate consequence. Instead, we propose strand-specific ssDNA gaps predominate and may later be converted into DSBs during late S/G2 processing, mitosis, or the next S phase. This review integrates mechanisms to connect structure dynamics with fork responses and downstream ssDNA gaps and breaks, providing possible models of structure-induced genome instability.

Abstract Highly conserved MutS and MutL homologs operate as protein dimers in mismatch repair (MMR). MutS recognizes mismatched nucleotides forming ATP-bound sliding clamps, which subsequently load MutL sliding clamps that coordinate MMR excision. Several MMR models envision static MutS-MutL complexes bound to mismatched DNA via a positively charged cleft (PCC) located on the MutL N-terminal domains (NTD). We show MutL-DNA binding is undetectable in physiological conditions. Instead, MutS sliding clamps exploit the PCC to position a MutL NTD on the DNA backbone, likely enabling diffusion-mediated wrapping of the remaining MutL domains around the DNA. The resulting MutL sliding clamp enhances MutH endonuclease and UvrD helicase activities on the DNA, which also engage the PCC during strand-specific incision/excision. These MutS clamp-loader progressions are significantly different from the replication clamp-loaders that attach the polymerase processivity factors β-clamp/PCNA to DNA, highlighting the breadth of mechanisms for stably linking crucial genome maintenance proteins onto DNA.

The accuracy by which organisms duplicate their DNA is of considerable interest. At least three mechanisms operate, serially, to secure high fidelity: base selection, exonucleolytic proofreading, and postreplicative mismatch correction. To obtain insights into the efficiency and specificity of these steps in the bacterium Escherichia coli, we have performed DNA sequence analysis of mutations occurring in the bacterial lacI gene in a series of strains genetically disabled in one or more of these error avoidance pathways. The base selection efficiency was estimated from mutagenesis occurring in a mutDmutL strain, which is deficient in both proofreading (mutD5) and mismatch repair (mutL). The proofreading efficiency was derived comparing the mutD5 mutL strain to the mismatch repair-deficient mutL strain. The efficiency of mismatch repair was derived comparing the mutL strain to the wild-type strain. The results show that base selection discriminates against errors by 200,000-2,000,000-fold, proofreading by 40-200-fold, and mismatch repair by 20-400-fold, each depending on the type of error. Base selection and proofreading act more strongly against transversions than transitions, whereas mismatch repair does the opposite. The data are based on 866 sequenced lacI mutations in a target that allows the scoring of at least 127 different mutations in 76 distinct DNA sequence contexts in vivo. They may therefore have general significance.

Highly conserved MutS homologs (MSH) and MutL homologs (MLH/PMS) are the fundamental components of mismatch repair (MMR). After decades of debate, it appears clear that the MSH proteins initiate MMR by recognizing a mismatch and forming multiple extremely stable ATP-bound sliding clamps that diffuse without hydrolysis along the adjacent DNA. The function(s) of MLH/PMS proteins is less clear, although they too bind ATP and are targeted to MMR by MSH sliding clamps. Structural analysis combined with recent real-time single molecule and cellular imaging technologies are providing new and detailed insight into the thermal-driven motions that animate the complete MMR mechanism.

Growing crystals of a DNA repair protein bound to DNA. This particular repair protein is the E. coli mismatch U:G DNA glycosylase. The crystals are produced for x-ray crystallographic studies to determine the structure of the protein-DNA complex. Biomedical Image Awards 2002.