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Many prokaryotes, including members of the Neisseria species, lack MutH and cannot employ methyl-directed DNA mismatch repair (MMR). The nick on the daughter strand is created by the endonuclease activity present in the C-terminal domain (CTD) of the MutL homodimer. MutL-CTD is known to interact with the processivity-clamp. The crystal structure of the homodimeric MutL-CTD from Neisseria (NgoL-CTD) in complex with homodimeric processivity-clamp (Nβ-Clamp) shows that each NgoL-CTD monomer binds to a Nβ-Clamp monomer through the conserved motif III (517QHLLIP522). The structure and allied biochemical studies plus in vivo growth assays conducted with wild-type (wt) plus mutant proteins shows that the endonuclease dimer sits transversely across the C-terminal face of the Nβ-Clamp ring. The comparison of the structure with that of the partial prokaryotic replisome suggests that the relative orientation of DNA, Nβ-Clamp, and NgoL-CTD may direct the daughter strand towards one of the active sites in endonuclease homodimer. Nicking assays conducted with wt and mutant NgoL-CTD in the presence and absence of Nβ-Clamp support this inference. Overall, our studies posit that strand discrimination in non-methyl-directed MMR is achieved through a structural strategy involving the β-Clamp which is distinct from the chemical strategy employed in prokaryotes like Escherichia coli., (© The Author(s) 2025. Published by Oxford University Press on behalf of Nucleic Acids Research.)

In eukaryotic post-replicative mismatch repair, MutS homolog complexes detect mismatches and in the major eukaryotic pathway, recruit Mlh1-Pms1/MLH1-PMS2 (yeast/human) complexes, which nick the newly replicated DNA strand upon activation by the replication processivity clamp, PCNA. This incision enables mismatch removal and DNA repair. Beyond its endonuclease role, Mlh1-Pms1/MLH1-PMS2 also has ATPase activity, which genetic studies suggest is essential for mismatch repair, although its precise regulatory role on DNA remains unclear. Here, we use an ATP-binding and hydrolysis-deficient yeast Mlh1-Pms1 variant to show that ATP hydrolysis promotes disengagement from Mlh1-Pms1-generated nicks, with hydrolysis in the Mlh1 subunit driving this activity. Our data suggest that the ATPase-deficient variant becomes trapped on its own endonuclease product, suggesting a mechanistic explanation for observations in genetic experiments. Additionally, we observed that Mlh1-Pms1 selectively protects DNA from exonuclease degradation at pre-existing nicks, which may act as strand discrimination signals in mismatch repair. Together, our findings suggest that Mlh1-Pms1 exhibits distinct behaviors on its own endonuclease products versus substrates with pre-existing nicks, supporting two distinct modes of action during DNA mismatch repair., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Fidelity of DNA replication is crucial for the accurate transmission of genetic information across generations, yet errors still occur despite multiple control mechanisms. This study investigated the factors influencing spontaneous replication errors across the Escherichia coli genome. We detected errors using the MutS and MutL mismatch repair proteins in rapidly proliferating mutH-deficient cells, where errors can be detected but not corrected. Our findings reveal that replication error hotspots are non-randomly distributed along the chromosome and are enriched in sequences with distinct features: lower thermal stability facilitating DNA strand separation, mononucleotide repeats prone to DNA polymerase slippage and sequences prone to forming secondary structures like cruciforms and G4 structures, which increase likelihood of DNA polymerase stalling. These hotspots showed enrichment for binding sites of nucleoid-associated proteins, RpoB and GyrA, as well as highly expressed genes, and depletion of GATC sequence. Finally, the enrichment of single-stranded DNA stretches in the hotspot regions establishes a nexus between the formation of secondary structures, transcriptional activity and replication stress. In conclusion, this study provides a comprehensive genome-wide map of replication error hotspots, offering a holistic perspective on the intricate interplay between various mechanisms that can compromise the faithful transmission of genetic information., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The accurate segregation of homologous chromosomes during the Meiosis I reductional division in most sexually reproducing eukaryotes requires crossing over between homologs. In baker's yeast approximately 80% of meiotic crossovers result from Mlh1-Mlh3 and Exo1 acting to resolve double-Holliday junction intermediates in a biased manner. Little is known about how Mlh1-Mlh3 is recruited to recombination intermediates to perform its role in crossover resolution. We performed a gene dosage screen in baker's yeast to identify novel genetic interactors with Mlh1-Mlh3. Specifically, we looked for genes whose lowered dosage reduced meiotic crossing over using sensitized mlh3 alleles that disrupt the stability of the Mlh1-Mlh3 complex and confer defects in mismatch repair but do not disrupt meiotic crossing over. To our surprise we identified genetic interactions between MLH3 and DMC1, the recombinase responsible for recombination between homologous chromosomes during meiosis. We then showed that Mlh3 physically interacts with Dmc1 in vitro and in vivo. Partial complementation of Mlh3 crossover functions was observed when MLH3 was expressed under the control of the CLB1 promoter (NDT80 regulon), suggesting that Mlh3 function can be provided late in meiotic prophase at some functional cost. A model for how Dmc1 could facilitate Mlh1-Mlh3's role in crossover resolution is presented., Competing Interests: Conflicts of interest The author declare no conflicts of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

The Repeat Expansion Diseases (REDs) arise from the expansion of a disease-specific short tandem repeat (STR). Different REDs differ with respect to the repeat involved, the cells that are most expansion prone and the extent of expansion. Furthermore, whether these diseases share a common expansion mechanism is unclear. To date, expansion has only been studied in a limited number of REDs. Here we report the first studies of the expansion mechanism in induced pluripotent stem cells derived from a patient with a form of the glutaminase deficiency disorder known as Global Developmental Delay, Progressive Ataxia, And Elevated Glutamine (GDPAG; OMIM# 618412) caused by the expansion of a CAG-STR in the 5' UTR of the glutaminase (GLS) gene. We show that alleles with as few as ~ 120 repeats show detectable expansions in culture despite relatively low levels of R-loops formed at this locus. Additionally, using a CRISPR-Cas9 knockout approach we show that PMS2 and MLH3, the constituents of MutLα and MutLγ, the 2 mammalian MutL complexes known to be involved in mismatch repair (MMR), are essential for expansion. Furthermore, PMS1, a component of a less well understood MutL complex, MutLβ, is also important, if not essential, for repeat expansion in these cells. Our results provide insights into the factors important for expansion and lend weight to the idea that, despite some differences, the same mechanism is responsible for expansion in many, if not all, REDs., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

The pathological huntingtin (HTT) trinucleotide repeat underlying Huntington disease (HD) continues to expand throughout life. Repeat length correlates both with earlier age at onset (AaO) and faster progression, making slowing its expansion an attractive therapeutic approach. Genome-wide association studies have identified candidate variants associated with altered AaO and progression, with many found in DNA mismatch repair (MMR)-associated genes. We examine whether lowering expression of these genes affects the rate of repeat expansion in human ex vivo models using HD iPSCs and HD iPSC-derived striatal medium spiny neuron-enriched cultures. We have generated a stable CRISPR interference HD iPSC line in which we can specifically and efficiently lower gene expression from a donor carrying over 125 CAG repeats. Lowering expression of each member of the MMR complexes MutS (MSH2, MSH3, and MSH6), MutL (MLH1, PMS1, PMS2, and MLH3), and LIG1 resulted in characteristic MMR deficiencies. Reduced MSH2, MSH3, and MLH1 slowed repeat expansion to the largest degree, while lowering either PMS1, PMS2, or MLH3 slowed it to a lesser degree. These effects were recapitulated in iPSC-derived striatal cultures where MutL factor expression was lowered. CRISPRi-mediated lowering of key MMR factor expression to levels feasibly achievable by current therapeutic approaches was able to effectively slow the expansion of the HTT CAG tract. We highlight members of the MutL family as potential targets to slow pathogenic repeat expansion with the aim to delay onset and progression of HD and potentially other repeat expansion disorders exhibiting somatic instability., Competing Interests: Declaration of interests In the past year, through the offices of UCL Consultants Ltd., a wholly owned subsidiary of University College London, S.J.T. has undertaken consultancy services for Alnylam Pharmaceuticals, Annexon, Ascidian Therapeutics, Arrowhead Pharmaceuticals, Atalanta Therapeutics, Design Therapeutics, F. Hoffman-La Roche, HCD Economics, IQVIA, Iris Medicine, Latus Bio, LifeEdit, Novartis Pharma, Pfizer, Prilenia Neurotherapeutics, PTC Therapeutics, Rgenta Therapeutics, Takeda Pharmaceuticals, UniQure Biopharma, and Vertex Pharmaceuticals. In the past 12 months, University College London Hospitals NHS Foundation Trust, S.J.T.’s host clinical institution, received funding to run clinical trials for F. Hoffman-La Roche, Novartis Pharma, PTC Therapeutics, and UniQure Biopharma., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The DNA mismatch repair (MMR) system promotes genome stability and protects humans from certain types of cancer. Its primary function is the correction of DNA polymerase errors. MutLα is an important eukaryotic MMR factor. We have examined the contributions of MutLα to maintaining genome stability. We show here that loss of MutLα in yeast increases the genome-wide mutation rate by ∼130-fold and generates a genome-wide mutation spectrum that consists of small indels and base substitutions. We also show that loss of yeast MutLα leads to error-prone MMR that produces T > C base substitutions in 5'-ATA-3' sequences. In agreement with this finding, our examination of human whole-genome DNA sequencing data has revealed that loss of MutLα in induced pluripotent stem cells triggers error-prone MMR that leads to the formation of T > C mutations in 5'-NTN-3' sequences. Our further analysis has shown that MutLα-independent MMR plays a role in suppressing base substitutions in N 3 homopolymeric runs. In addition, we describe that MutLα preferentially protects noncoding DNA from mutations. Our study defines the contributions of MutLα-dependent and independent mechanisms to genome-wide MMR., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

MutL family proteins contain an N-terminal ATPase domain (NTD), an unstructured interdomain linker, and a C-terminal domain (CTD), which mediates constitutive dimerization between subunits and often contains an endonuclease active site. Most MutL homologs direct strand-specific DNA mismatch repair by cleaving the error-containing daughter DNA strand. The strand cleavage reaction is poorly understood; however, the structure of the endonuclease active site is consistent with a two- or three-metal ion cleavage mechanism. A motif required for this endonuclease activity is present in the unstructured linker of Mlh1 and is conserved in all eukaryotic Mlh1 proteins, except those from metamonads, which also lack the almost absolutely conserved Mlh1 C-terminal phenylalanine-glutamate-arginine-cysteine (FERC) sequence. We hypothesize that the cysteine in the FERC sequence is autoinhibitory, as it sequesters the active site. We further hypothesize that the evolutionary co-occurrence of the conserved linker motif with the FERC sequence indicates a functional interaction, possibly by linker motif-mediated displacement of the inhibitory cysteine. This role is consistent with available data for interactions between the linker motif with DNA and the CTDs in the vicinity of the active site., (© 2023 The Authors. BioEssays published by Wiley Periodicals LLC.)

MutLα is essential for human DNA mismatch repair (MMR). It harbors a latent endonuclease, is responsible for recruitment of process associated proteins and is relevant for strand discrimination. Recently, we demonstrated that the MMR function of MutLα is regulated by phosphorylation of MLH1 at serine (S) 477. In the current study, we focused on S87 located in the ATPase domain of MLH1 and on S446, S456 and S477 located in its linker region. We analysed the phosphorylation-dependent impact of these amino acids on DNA binding, MMR ability and thermal stability of MutLα. We were able to demonstrate that phosphorylation at S87 of MLH1 inhibits DNA binding of MutLα. In addition, we detected that its MMR function seems to be regulated predominantly via phosphorylation of serines in the linker domain, which are also partially involved in the regulation of DNA binding. Furthermore, we found that the thermal stability of MutLα decreased in relation to its phosphorylation status implying that complete phosphorylation might lead to instability and degradation of MLH1. In summary, we showed here, for the first time, a phosphorylation-dependent regulation of DNA binding of MutLα and hypothesized that this might significantly impact its functional regulation during MMR in vivo., (© 2023. The Author(s).)

In eukaryotic mismatch repair, MutS homologs recognize mismatches and recruit the MutLα endonuclease which introduces a nick in the newly replicated, error-containing DNA strand. The nick occurs in response to the mismatch, but at a site up to several hundred base pairs away. The MutLα nick promotes mismatch excision by an exonuclease (Exo1) or removal by the strand displacement activity of a DNA polymerase which may work in conjunction with a flap endonuclease. Models have suggested that MutL homolog endonucleases form oligomeric complexes which facilitate and are activated by strand capture mechanisms, although such models have never been explicitly tested. We present evidence that the mismatch repair MutLα endonuclease is activated by DNA-DNA associations and that it can use this property to overcome DNA torsional barriers. Using DNA ligation and pull-down experiments, we determined that the MutLα endonuclease associates two DNA duplexes. Using nuclease assays, we determined that this activity stimulates MutLα's endonuclease function. We also observe that MutLα enhances a topoisomerase without nicking the DNA itself. Our data provide a mechanistic explanation for how MutL proteins interact with DNA during mismatch repair, and how MutL homologs participate in other processes, such as recombination and trinucleotide repeat expansions., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Eukaryotic DNA mismatch repair (MMR) initiates through mispair recognition by the MutS homologs Msh2-Msh6 and Msh2-Msh3 and subsequent recruitment of the MutL homologs Mlh1-Pms1 (human MLH1-PMS2). In bacteria, MutL is recruited by interactions with the connector domain of one MutS subunit and the ATPase and core domains of the other MutS subunit. Analysis of the S. cerevisiae and human homologs have only identified an interaction between the Msh2 connector domain and Mlh1. Here we investigated whether a conserved Msh6 ATPase/core domain-Mlh1 interaction and an Msh2-Msh6 interaction with Pms1 also act in MMR. Mutations in MLH1 affecting interactions with both the Msh2 and Msh6 interfaces caused MMR defects, whereas equivalent pms1 mutations did not cause MMR defects. Mutant Mlh1-Pms1 complexes containing Mlh1 amino acid substitutions were defective for recruitment to mispaired DNA by Msh2-Msh6, did not support MMR in reconstituted Mlh1-Pms1-dependent MMR reactions in vitro, but were proficient in Msh2-Msh6-independent Mlh1-Pms1 endonuclease activity. These results indicate that Mlh1, the common subunit of the Mlh1-Pms1, Mlh1-Mlh2, and Mlh1-Mlh3 complexes, but not Pms1, is recruited by Msh2-Msh6 through interactions with both of its subunits., Competing Interests: Conflict of interest The authors declare that they have no conflict of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

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., (© 2022. The Author(s).)

Neisseria gonorrhoeae (a Gram-negative diplococcus) is a human pathogen and causative agent of gonorrhea, a sexually transmitted infection. The bacterium uses various approaches for adapting to environmental conditions and multiplying efficiently in the human body, such as regulation of expression of gene expression of surface proteins and lipooligosaccharides (e.g., expression of various forms of pilin). The systems of DNA repair play an important role in the bacterium ability to survive in the host body. This review describes DNA repair systems of N. gonorrhoeae and their role in the pathogenicity of this bacterium. A special attention is paid to the mismatch repair system (MMR) and functioning of the MutS and MutL proteins, as well as to the role of these proteins in regulation of the pilin antigenic variation of the N. gonorrhoeae pathogen.

In eukaryotes, the origin recognition complex (ORC) is required for the initiation of DNA replication. The smallest subunit of ORC, Orc6, is essential for prereplication complex (pre-RC) assembly and cell viability in yeast and for cytokinesis in metazoans. However, unlike other ORC components, the role of human Orc6 in replication remains to be resolved. Here, we identify an unexpected role for hOrc6, which is to promote S-phase progression after pre-RC assembly and DNA damage response. Orc6 localizes at the replication fork and is an accessory factor of the mismatch repair (MMR) complex. In response to oxidative damage during S phase, often repaired by MMR, Orc6 facilitates MMR complex assembly and activity, without which the checkpoint signaling is abrogated. Mechanistically, Orc6 directly binds to MutSα and enhances the chromatin-association of MutLα, thus enabling efficient MMR. Based on this, we conclude that hOrc6 plays a fundamental role in genome surveillance during S phase.

Eukaryotic DNA Mismatch Repair (MMR) involves redundant exonuclease 1 (Exo1)-dependent and Exo1-independent pathways, of which the Exo1-independent pathway(s) is not well understood. The exo1Δ440-702 mutation, which deletes the MutS Homolog 2 (Msh2) and MutL Homolog 1 (Mlh1) interacting peptides (SHIP and MIP boxes, respectively), eliminates the Exo1 MMR functions but is not lethal in combination with rad27Δ mutations. Analyzing the effect of different combinations of the exo1Δ440-702 mutation, a rad27Δ mutation and the pms1-A99V mutation, which inactivates an Exo1-independent MMR pathway, demonstrated that each of these mutations inactivates a different MMR pathway. Furthermore, it was possible to reconstitute a Rad27- and Msh2-Msh6-dependent MMR reaction in vitro using a mispaired DNA substrate and other MMR proteins. Our results demonstrate Rad27 defines an Exo1-independent eukaryotic MMR pathway that is redundant with at least two other MMR pathways., (© 2021. The Author(s).)

Embryonic aneuploidy from mis-segregation of chromosomes during meiosis causes pregnancy loss. Proper disjunction of homologous chromosomes requires the mismatch repair (MMR) genes MLH1 and MLH3, essential in mice for fertility. Variants in these genes can increase colorectal cancer risk, yet the reproductive impacts are unclear. To determine if MLH1/3 single nucleotide polymorphisms (SNPs) in human populations could cause reproductive abnormalities, we use computational predictions, yeast two-hybrid assays, and MMR and recombination assays in yeast, selecting nine MLH1 and MLH3 variants to model in mice via genome editing. We identify seven alleles causing reproductive defects in mice including female subfertility and male infertility. Remarkably, in females these alleles cause age-dependent decreases in litter size and increased embryo resorption, likely a consequence of fewer chiasmata that increase univalents at meiotic metaphase I. Our data suggest that hypomorphic alleles of meiotic recombination genes can predispose females to increased incidence of pregnancy loss from gamete aneuploidy., (© 2021. The Author(s).)

Meiotic recombination is essential for reciprocal exchange of genetic information between homologous chromosomes and their subsequent proper segregation in sexually reproducing organisms. MLH1 and MLH3 belong to meiosis-specific members of the MutL-homolog family, which are required for normal level of crossovers (COs) in some eukaryotes. However, their functions in plants need to be further elucidated. Here, we report the identification of OsMLH1 and reveal its functions during meiosis in rice. Using CRISPR-Cas9 approach, two independent mutants, Osmlh1-1 and Osmlh1-2, are generated and exhibited significantly reduced male fertility. In Osmlh1-1, the clearance of PAIR2 is delayed and partial ZEP1 proteins are not loaded into the chromosomes, which might be due to the deficient in resolution of interlocks at late zygotene. Thus, OsMLH1 is required for the assembly of synapsis complex. In Osmlh1-1, CO number is dropped by ~53% and the distribution of residual COs is consistent with predicted Poisson distribution, indicating that OsMLH1 is essential for the formation of interference-sensitive COs (class I COs). OsMLH1 interacts with OsMLH3 through their C-terminal domains. Mutation in OsMLH3 also affects the pollen fertility. Thus, our experiments reveal that the conserved heterodimer MutLγ (OsMLH1-OsMLH3) is essential for the formation of class I COs in rice., Competing Interests: Conflict of interest The authors have declared no competing interests., (Copyright © 2021 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Ltd. All rights reserved.)

Baker's yeast contains a large number of duplicated genes; some function redundantly, whereas others have more specialized roles. We used the MLH family of DNA mismatch repair (MMR) proteins as a model to better understand the steps that lead to gene specialization following a gene duplication event. We focused on two highly conserved yeast MLH proteins, Pms1 and Mlh3, with Pms1 having a major role in the repair of misincorporation events during DNA replication and Mlh3 acting to resolve recombination intermediates in meiosis to form crossovers. The baker's yeast Mlh3 and Pms1 proteins are significantly diverged (19% overall identity), suggesting that an extensive number of evolutionary steps, some major, others involving subtle refinements, took place to diversify the MLH proteins. Using phylogenetic and molecular approaches, we provide evidence that all three domains (N-terminal ATP binding, linker, C-terminal endonuclease/MLH interaction) in the MLH protein family are critical for conferring pathway specificity. Importantly, mlh3 alleles in the ATP binding and endonuclease domains improved MMR functions in strains lacking the Pms1 protein and did not disrupt Mlh3 meiotic functions. This ability for mlh3 alleles to complement the loss of Pms1 suggests that an ancestral Pms1/Mlh3 protein was capable of performing both MMR and crossover functions. Our strategy for analyzing MLH pathway specificity provides an approach to understand how paralogs have evolved to support distinct cellular processes., (© The Author(s) 2021. Published by Oxford University Press on behalf of Genetics Society of America.)

In budding yeast, the MutL homolog heterodimer Mlh1-Mlh3 (MutLγ) plays a central role in the formation of meiotic crossovers. It is also involved in the repair of a subset of mismatches besides the main mismatch repair (MMR) endonuclease Mlh1-Pms1 (MutLα). The heterodimer interface and endonuclease sites of MutLγ and MutLα are located in their C-terminal domain (CTD). The molecular basis of MutLγ's dual roles in MMR and meiosis is not known. To better understand the specificity of MutLγ, we characterized the crystal structure of Saccharomyces cerevisiae MutLγ(CTD). Although MutLγ(CTD) presents overall similarities with MutLα(CTD), it harbors some rearrangement of the surface surrounding the active site, which indicates altered substrate preference. The last amino acids of Mlh1 participate in the Mlh3 endonuclease site as previously reported for Pms1. We characterized mlh1 alleles and showed a critical role of this Mlh1 extreme C terminus both in MMR and in meiotic recombination. We showed that the MutLγ(CTD) preferentially binds Holliday junctions, contrary to MutLα(CTD). We characterized Mlh3 positions on the N-terminal domain (NTD) and CTD that could contribute to the positioning of the NTD close to the CTD in the context of the full-length MutLγ. Finally, crystal packing revealed an assembly of MutLγ(CTD) molecules in filament structures. Mutation at the corresponding interfaces reduced crossover formation, suggesting that these superstructures may contribute to the oligomer formation proposed for MutLγ. This study defines clear divergent features between the MutL homologs and identifies, at the molecular level, their specialization toward MMR or meiotic recombination functions., Competing Interests: The authors declare no competing interest.

Resistance to endocrine treatment occurs in ~30% of ER + breast cancer patients resulting in ~40,000 deaths/year in the USA. Preclinical studies strongly implicate activation of growth factor receptor, HER2 in endocrine treatment resistance. However, clinical trials of pan-HER inhibitors in ER + /HER2 - patients have disappointed, likely due to a lack of predictive biomarkers. Here we demonstrate that loss of mismatch repair activates HER2 after endocrine treatment in ER + /HER2 - breast cancer cells by protecting HER2 from protein trafficking. Additionally, HER2 activation is indispensable for endocrine treatment resistance in MutL - cells. Consequently, inhibiting HER2 restores sensitivity to endocrine treatment. Patient data from multiple clinical datasets supports an association between MutL loss, HER2 upregulation, and sensitivity to HER inhibitors in ER + /HER2 - patients. These results provide strong rationale for MutL loss as a first-in-class predictive marker of sensitivity to combinatorial treatment with endocrine intervention and HER inhibitors in endocrine treatment-resistant ER + /HER2 - breast cancer patients.

Meiotic recombination ensures proper chromosome segregation to form viable gametes and results in gene conversions events between homologs. Conversion tracts are shorter in meiosis than in mitotically dividing cells. This results at least in part from the binding of a complex, containing the Mer3 helicase and the MutLβ heterodimer, to meiotic recombination intermediates. The molecular actors inhibited by this complex are elusive. The Pif1 DNA helicase is known to stimulate DNA polymerase delta (Pol δ) -mediated DNA synthesis from D-loops, allowing long synthesis required for break-induced replication. We show that Pif1 is also recruited genome wide to meiotic DNA double-strand break (DSB) sites. We further show that Pif1, through its interaction with PCNA, is required for the long gene conversions observed in the absence of MutLβ recruitment to recombination sites. In vivo, Mer3 interacts with the PCNA clamp loader RFC, and in vitro, Mer3-MutLβ ensemble inhibits Pif1-stimulated D-loop extension by Pol δ and RFC-PCNA. Mechanistically, our results suggest that Mer3-MutLβ may compete with Pif1 for binding to RFC-PCNA. Taken together, our data show that Pif1's activity that promotes meiotic DNA repair synthesis is restrained by the Mer3-MutLβ ensemble which in turn prevents long gene conversion tracts and possibly associated mutagenesis., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The MutL family of DNA mismatch repair proteins (MMR) acts to maintain genomic integrity in somatic and meiotic cells. In baker's yeast, the MutL homolog (MLH) MMR proteins form three heterodimeric complexes, MLH1-PMS1, MLH1-MLH2, and MLH1-MLH3. The recent discovery of human PMS2 (homolog of baker's yeast PMS1) and MLH3 acting independently of human MLH1 in the repair of somatic double-strand breaks questions the assumption that MLH1 is an obligate subunit for MLH function. Here we provide a summary of the canonical roles for MLH factors in DNA genomic maintenance and in meiotic crossover. We then present the phenotypes of cells lacking specific MLH subunits, particularly in meiotic recombination, and based on this analysis, propose a model for an independent early role for MLH3 in meiosis to promote the accurate segregation of homologous chromosomes in the meiosis I division.

The DNA mismatch repair (MMR) process detects and corrects replication errors in organisms ranging from bacteria to humans. In most bacteria, it is initiated by MutS detecting mismatches and MutL nicking the mismatch-containing DNA strand. Here, we show that MMR reduces the appearance of rifampicin resistances more than a 100-fold in the Caulobacter crescentus Alphaproteobacterium. Using fluorescently-tagged and functional MutS and MutL proteins, live cell microscopy experiments showed that MutS is usually associated with the replisome during the whole S-phase of the C. crescentus cell cycle, while MutL molecules may display a more dynamic association with the replisome. Thus, MMR components appear to use a 1D-scanning mode to search for rare mismatches, although the spatial association between MutS and the replisome is dispensible under standard growth conditions. Conversely, the spatial association of MutL with the replisome appears as critical for MMR in C. crescentus, suggesting a model where the β-sliding clamp licences the endonuclease activity of MutL right behind the replication fork where mismatches are generated. The spatial association between MMR and replisome components may also play a role in speeding up MMR and/or in recognizing which strand needs to be repaired in a variety of Alphaproteobacteria., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The mismatch repair system (MMR) ensures the stability of genetic information during DNA replication in almost all organisms. Mismatch repair is initiated after recognition of a non-canonical nucleotide pair by the MutS protein and the formation of a complex between MutS and MutL. Eukaryotic and most bacterial MutL homologs function as endonucleases that introduce a single-strand break in the daughter strand of the DNA, thus activating the repair process. However, many aspects of the functioning of this protein remain unknown. We studied the ATPase and DNA binding functions of the MutL protein from the pathogenic bacterium Neisseria gonorrhoeae (NgoMutL), which exhibits endonuclease activity. For the first time, the kinetic parameters of ATP hydrolysis by the full-length NgoMutL protein were determined. Its interactions with single- and double-stranded DNA fragments of various lengths were studied. NgoMutL was shown to be able to efficiently form complexes with DNA fragments that are longer than 40 nucleotides. Using modified DNA duplexes harboring a 2-pyridyldisulfide group on linkers of various lengths, we obtained NgoMutL conjugates with DNA for the first time. According to these results, the Cys residues of the wild-type protein are located at a distance of approximately 18-50 Å from the duplex. The efficiency of the affinity modification of Cys residues in NgoMutL with reactive DNAs was shown to decrease in the presence of ATP or its non-hydrolyzable analog, as well as ZnCl2, in the reaction mixture. We hypothesize that the conserved Cys residues of the C-terminal domain of NgoMutL, which are responsible for the coordination of metal ions in the active center of the protein, are involved in its interaction with DNA. This information may be useful in reconstruction of the main stages of MMR in prokaryotes that are different from γ-proteobacteria, as well as in the search for new targets for drugs against N. gonorrhoeae.

The MutL family of DNA mismatch repair proteins plays a critical role in excising and repairing misincorporation errors during DNA replication. In many eukaryotes, members of this family have evolved to modulate and resolve recombination intermediates into crossovers during meiosis. In these organisms, such functions promote the accurate segregation of chromosomes during the meiosis I division. What alterations occurred in MutL homolog (MLH) family members that enabled them to acquire these new roles? In this review, we present evidence that the yeast Mlh1-Mlh3 and Mlh1-Mlh2 complexes have evolved novel enzymatic and nonenzymatic activities and protein-protein interactions that are critical for their meiotic functions. Curiously, even with these changes, these complexes retain backup and accessory roles in DNA mismatch repair during vegetative growth., (© 2020 John Wiley & Sons, Ltd.)

Homologous recombination (HR) repairs DNA double-strand breaks using intact homologous sequences as template DNA. Broken DNA and intact homologous sequences form joint molecules (JMs), including Holliday junctions (HJs), as HR intermediates. HJs are resolved to form crossover and noncrossover products. A mismatch repair factor, MLH3 endonuclease, produces the majority of crossovers during meiotic HR, but it remains elusive whether mismatch repair factors promote HR in nonmeiotic cells. We disrupted genes encoding the MLH3 and PMS2 endonucleases in the human B cell line, TK6, generating null MLH3 -/- and PMS2 -/- mutant cells. We also inserted point mutations into the endonuclease motif of MLH3 and PMS2 genes, generating endonuclease death MLH3 DN/DN and PMS2 EK/EK cells. MLH3 -/- and MLH3 DN/DN cells showed a very similar phenotype, a 2.5-fold decrease in the frequency of heteroallelic HR-dependent repair of restriction enzyme-induced double-strand breaks. PMS2 -/- and PMS2 EK/EK cells showed a phenotype very similar to that of the MLH3 mutants. These data indicate that MLH3 and PMS2 promote HR as an endonuclease. The MLH3 DN/DN and PMS2 EK/EK mutations had an additive effect on the heteroallelic HR. MLH3 DN/DN /PMS2 EK/EK cells showed normal kinetics of γ-irradiation-induced Rad51 foci but a significant delay in the resolution of Rad51 foci and a 3-fold decrease in the number of cisplatin-induced sister chromatid exchanges. The ectopic expression of the Gen1 HJ re-solvase partially reversed the defective heteroallelic HR of MLH3 DN/DN /PMS2 EK/EK cells. Taken together, we propose that MLH3 and PMS2 promote HR as endonucleases, most likely by processing JMs in mammalian somatic cells., (Copyright © 2020 © 2020 Rahman et al. Published by Elsevier Inc. All rights reserved.)

Inactivating mutations affecting key mismatch repair (MMR) components lead to microsatellite instability (MSI) and cancer. However, a number of patients with MSI-tumors do not present alterations in classical MMR genes. Here we discovered that specific missense mutations in the MutL homolog MLH2, which is dispensable for MMR, confer a dominant mutator phenotype in S. cerevisiae. MLH2 mutations elevated frameshift mutation rates, and caused accumulation of long-lasting nuclear MMR foci. Both aspects of this phenotype were suppressed by mutations predicted to prevent the binding of Mlh2 to DNA. Genetic analysis revealed that mlh2 dominant mutations interfere with both Exonuclease 1 (Exo1)-dependent and Exo1-independent MMR. Lastly, we demonstrate that a homolog mutation in human hPMS1 results in a dominant mutator phenotype. Our data support a model in which yeast Mlh1-Mlh2 or hMLH1-hPMS1 mutant complexes act as roadblocks on DNA preventing MMR, unraveling a novel mechanism that can account for MSI in human cancer.

Crossovers generated during the repair of programmed meiotic double-strand breaks must be tightly regulated to promote accurate homolog segregation without deleterious outcomes, such as aneuploidy. The Mlh1-Mlh3 (MutLγ) endonuclease complex is critical for crossover resolution, which involves mechanistically unclear interplay between MutLγ and Exo1 and polo kinase Cdc5. Using budding yeast to gain temporal and genetic traction on crossover regulation, we find that MutLγ constitutively interacts with Exo1. Upon commitment to crossover repair, MutLγ-Exo1 associate with recombination intermediates, followed by direct Cdc5 recruitment that triggers MutLγ crossover activity. We propose that Exo1 serves as a central coordinator in this molecular interplay, providing a defined order of interaction that prevents deleterious, premature activation of crossovers. MutLγ associates at a lower frequency near centromeres, indicating that spatial regulation across chromosomal regions reduces risky crossover events. Our data elucidate the temporal and spatial control surrounding a constitutive, potentially harmful, nuclease. We also reveal a critical, noncatalytic role for Exo1, through noncanonical interaction with polo kinase. These mechanisms regulating meiotic crossovers may be conserved across species., Competing Interests: The authors declare no competing interest.

DNA mismatch repair (MMR) plays a crucial role in the maintenance of genomic stability. The main MMR protein, MutS, was recently shown to recognize the G-quadruplex (G4) DNA structures, which, along with regulatory functions, have a negative impact on genome integrity. Here, we studied the effect of G4 on the DNA-binding activity of MutS from Rhodobacter sphaeroides (methyl-independent MMR) in comparison with MutS from Escherichia coli (methyl-directed MMR) and evaluated the influence of a G4 on the functioning of other proteins involved in the initial steps of MMR. For this purpose, a new DNA construct was designed containing a biologically relevant intramolecular stable G4 structure flanked by double-stranded regions with the set of DNA sites required for MMR initiation. The secondary structure of this model was examined using NMR spectroscopy, chemical probing, fluorescent indicators, circular dichroism, and UV spectroscopy. The results unambiguously showed that the d(GGGT) 4 motif, when embedded in a double-stranded context, adopts a G4 structure of a parallel topology. Despite strong binding affinities of MutS and MutL for a G4, the latter is not recognized by E. coli MMR as a signal for repair, but does not prevent MMR processing when a G4 and G/T mismatch are in close proximity.

During prophase of the first meiotic division, cells deliberately break their DNA 1 . These DNA breaks are repaired by homologous recombination, which facilitates proper chromosome segregation and enables the reciprocal exchange of DNA segments between homologous chromosomes 2 . A pathway that depends on the MLH1-MLH3 (MutLγ) nuclease has been implicated in the biased processing of meiotic recombination intermediates into crossovers by an unknown mechanism 3-7 . Here we have biochemically reconstituted key elements of this pro-crossover pathway. We show that human MSH4-MSH5 (MutSγ), which supports crossing over 8 , binds branched recombination intermediates and associates with MutLγ, stabilizing the ensemble at joint molecule structures and adjacent double-stranded DNA. MutSγ directly stimulates DNA cleavage by the MutLγ endonuclease. MutLγ activity is further stimulated by EXO1, but only when MutSγ is present. Replication factor C (RFC) and the proliferating cell nuclear antigen (PCNA) are additional components of the nuclease ensemble, thereby triggering crossing-over. Saccharomyces cerevisiae strains in which MutLγ cannot interact with PCNA present defects in forming crossovers. Finally, the MutLγ-MutSγ-EXO1-RFC-PCNA nuclease ensemble preferentially cleaves DNA with Holliday junctions, but shows no canonical resolvase activity. Instead, it probably processes meiotic recombination intermediates by nicking double-stranded DNA adjacent to the junction points 9 . As DNA nicking by MutLγ depends on its co-factors, the asymmetric distribution of MutSγ and RFC-PCNA on meiotic recombination intermediates may drive biased DNA cleavage. This mode of MutLγ nuclease activation might explain crossover-specific processing of Holliday junctions or their precursors in meiotic chromosomes 4 .

During meiosis, crossover recombination connects homologous chromosomes to direct their accurate segregation 1 . Defective crossing over causes infertility, miscarriage and congenital disease. Each pair of chromosomes attains at least one crossover via the formation and biased resolution of recombination intermediates known as double Holliday junctions 2,3 . A central principle of crossover resolution is that the two Holliday junctions are resolved in opposite planes by targeting nuclease incisions to specific DNA strands 4 . The endonuclease activity of the MutLγ complex has been implicated in the resolution of crossovers 5-10 , but the mechanisms that activate and direct strand-specific cleavage remain unknown. Here we show that the sliding clamp PCNA is important for crossover-biased resolution. In vitro assays with human enzymes show that PCNA and its loader RFC are sufficient to activate the MutLγ endonuclease. MutLγ is further stimulated by a co-dependent activity of the pro-crossover factors EXO1 and MutSγ, the latter of which binds Holliday junctions 11 . MutLγ also binds various branched DNAs, including Holliday junctions, but does not show canonical resolvase activity, implying that the endonuclease incises adjacent to junction branch points to achieve resolution. In vivo, RFC facilitates MutLγ-dependent crossing over in budding yeast. Furthermore, PCNA localizes to prospective crossover sites along synapsed chromosomes. These data highlight similarities between crossover resolution and the initiation steps of DNA mismatch repair 12,13 and evoke a novel model for crossover-specific resolution of double Holliday junctions during meiosis.

DNA mismatch repair (MMR), the guardian of the genome, commences when MutS identifies a mismatch and recruits MutL to nick the error-containing strand, allowing excision and DNA resynthesis. Dominant MMR models posit that after mismatch recognition, ATP converts MutS to a hydrolysis-independent, diffusive mobile clamp that no longer recognizes the mismatch. Little is known about the postrecognition MutS mobile clamp and its interactions with MutL. Two disparate frameworks have been proposed: One in which MutS-MutL complexes remain mobile on the DNA, and one in which MutL stops MutS movement. Here we use single-molecule FRET to follow the postrecognition states of MutS and the impact of MutL on its properties. In contrast to current thinking, we find that after the initial mobile clamp formation event, MutS undergoes frequent cycles of mismatch rebinding and mobile clamp reformation without releasing DNA. Notably, ATP hydrolysis is required to alter the conformation of MutS such that it can recognize the mismatch again instead of bypassing it; thus, ATP hydrolysis licenses the MutS mobile clamp to rebind the mismatch. Moreover, interaction with MutL can both trap MutS at the mismatch en route to mobile clamp formation and stop movement of the mobile clamp on DNA. MutS's frequent rebinding of the mismatch, which increases its residence time in the vicinity of the mismatch, coupled with MutL's ability to trap MutS, should increase the probability that MutS-MutL MMR initiation complexes localize near the mismatch., Competing Interests: The authors declare no competing interest.

DNA mismatch repair (MMR) corrects errors that occur during DNA replication. In humans, mutations in the proteins MutSα and MutLα that initiate MMR cause Lynch syndrome, the most common hereditary cancer. MutSα surveilles the DNA, and upon recognition of a replication error it undergoes adenosine triphosphate-dependent conformational changes and recruits MutLα. Subsequently, proliferating cell nuclear antigen (PCNA) activates MutLα to nick the error-containing strand to allow excision and resynthesis. The structure-function properties of these obligate MutSα-MutLα complexes remain mostly unexplored in higher eukaryotes, and models are predominately based on studies of prokaryotic proteins. Here, we utilize atomic force microscopy (AFM) coupled with other methods to reveal time- and concentration-dependent stoichiometries and conformations of assembling human MutSα-MutLα-DNA complexes. We find that they assemble into multimeric complexes comprising three to eight proteins around a mismatch on DNA. On the timescale of a few minutes, these complexes rearrange, folding and compacting the DNA. These observations contrast with dominant models of MMR initiation that envision diffusive MutS-MutL complexes that move away from the mismatch. Our results suggest MutSα localizes MutLα near the mismatch and promotes DNA configurations that could enhance MMR efficiency by facilitating MutLα nicking the DNA at multiple sites around the mismatch. In addition, such complexes may also protect the mismatch region from nucleosome reassembly until repair occurs, and they could potentially remodel adjacent nucleosomes., Competing Interests: The authors declare no competing interest.

Expansion of a CGG-repeat tract in the 5' untranslated region of the FMR1 gene causes the fragile X-related disorders (FXDs; aka the FMR1 disorders). The expansion mechanism is likely shared by the 35+ other diseases resulting from expansion of a disease-specific microsatellite, but many steps in this process are unknown. We have shown previously that expansion is dependent upon functional mismatch repair proteins, including an absolute requirement for MutLγ, one of the three MutL heterodimeric complexes found in mammalian cells. We demonstrate here that both MutLα and MutLβ, the two other MutL complexes present in mammalian cells, are also required for most, if not all, expansions in a mouse embryonic stem cell model of the FXDs. A role for MutLα and MutLβ is consistent with human GWA studies implicating these complexes as modifiers of expansion risk in other Repeat Expansion Diseases. The requirement for all three complexes suggests a novel model in which these complexes co-operate to generate expansions. It also suggests that the PMS1 subunit of MutLβ may be a reasonable therapeutic target in those diseases in which somatic expansion is an important disease modifier., Competing Interests: The authors have declared that no competing interests exist.

In this retrospective study, we evaluated the predictive role of different immunohistochemical expression (IHC) of the mismatch repair proteins (MMR) in patients with colorectal liver metastasis (CRLM) submitted to liver resection. A total of 108 patients were retrieved, and 15 patients were excluded from the study because of the impossibility to obtain adequate formalin-fixed tissue blocks. The final analysis included 93 patients. Twenty-eight cases (30%) presented a no loss of expression or microsatellite stability (MSS) status, 59 cases (63%) showed a hybrid loss of expression, while 6 cases (7%) presented a complete loss of expression or microsatellite instability status (MSI). Patients with complete or hybrid loss of expression of MMR developed a high intra-hepatic recurrence rate compared to other ones (54% vs 21% OR of 4.278, 95% CI 1.53-11.93) (p = 0.004). The same difference in terms of liver recurrence has been found among patients with R0 resection (50% vs 17% OR of 0.2, 95% CI 0.06-0.65) (p = 0.005). However, there was no difference in terms of disease-free survival and overall survival. Complete or hybrid loss of expression of MMR could be considered a risk factor for intra-hepatic recurrences after liver resections for CRLM.

MutL proteins are ubiquitous and play important roles in DNA metabolism. MutLγ (MLH1-MLH3 heterodimer) is a poorly understood member of the eukaryotic family of MutL proteins that has been implicated in triplet repeat expansion, but its action in this deleterious process has remained unknown. In humans, triplet repeat expansion is the molecular basis for ∼40 neurological disorders. In addition to MutLγ, triplet repeat expansion involves the mismatch recognition factor MutSβ (MSH2-MSH3 heterodimer). We show here that human MutLγ is an endonuclease that nicks DNA. Strikingly, incision of covalently closed, relaxed loop-containing DNA by human MutLγ is promoted by MutSβ and targeted to the strand opposite the loop. The resulting strand break licenses downstream events that lead to a DNA expansion event in human cell extracts. Our data imply that the mammalian MutLγ is a unique endonuclease that can initiate triplet repeat DNA expansions., Competing Interests: Competing interest statement: P.L.M. serves on the Scientific Advisory Board of Elysium Health., (Copyright © 2020 the Author(s). Published by PNAS.)

The mismatch repair (MMR) complex is composed of MutSα (MSH2-MSH6) and MutLα (MLH1-PMS2) and specifically recognizes mismatched bases during DNA replication. O 6 -Methylguanine is produced by treatment with alkylating agents, such as N -methyl- N -nitrosourea (MNU), and during DNA replication forms a DNA mismatch ( i.e. an O 6 -methylguanine/thymine pair) and induces a G/C to A/T transition mutation. To prevent this outcome, cells carrying this DNA mismatch are eliminated by MMR-dependent apoptosis, but the underlying molecular mechanism is unclear. In this study, we provide evidence that the chromatin-regulatory and ATP-dependent nucleosome-remodeling protein SMARCAD1 is involved in the induction of MMR-dependent apoptosis in human cells. Unlike control cells, SMARCAD1 -knockout cells (ΔSMARCAD1) were MNU-resistant, and the appearance of a sub-G 1 population and caspase-9 activation were significantly suppressed in the ΔSMARCAD1 cells. Furthermore, the MNU-induced mutation frequencies were increased in these cells. Immunoprecipitation analyses revealed that the recruitment of MutLα to chromatin-bound MutSα, observed in SMARCAD1-proficient cells, is suppressed in ΔSMARCAD1 cells. Of note, the effect of SMARCAD1 on the recruitment of MutLα exclusively depended on the ATPase activity of the protein. On the basis of these findings, we propose that SMARCAD1 induces apoptosis via its chromatin-remodeling activity, which helps recruit MutLα to MutSα on damaged chromatin., (© 2020 Takeishi et al.)

DNA mismatch repair (MMR) increases replication fidelity and genome stability by correcting DNA polymerase errors that remain after replication. Defects in MMR result in the accumulation of mutations and lead to human tumor development. Germline mutations in MMR cause the hereditary cancer syndrome, Lynch syndrome. After replication, DNA is reorganized into its chromatin structure and wrapped around histone octamers. DNA MMR is thought to be less efficient in recognizing and repairing mispairs packaged in chromatin, in which case MMR must either compete for access to naked DNA before histone deposition or actively move nucleosomes to access the mispair. This article reviews studies into the mechanistic and physical interactions between MMR and various chromatin-associated factors, including the histone deposition complex CAF1. Recent Xenopus and Saccharomyces cerevisiae studies describe a physical interaction between Msh2 and chromatin-remodeling ATPase Fun30/SMARCAD1, with potential mechanistic roles for SMARCAD1 in moving histones for both mispair access and excision tract elongation. The RSC complex, another histone remodeling complex, also potentially influences excision tract length. Deletion mutations of RSC2 point to mechanistic interactions with the MMR pathways. Together, these studies paint a picture of complex interactions between MMR and the chromatin environment that will require numerous additional genetic, biochemical, and cell biology experiments to fully understand. Understanding how these pathways interconnect is essential in fully understanding eukaryotic MMR and has numerous implications in human tumor formation and treatment., (Copyright © 2019 Elsevier B.V. All rights reserved.)

DNA mismatch repair (MMR) maintains genome stability through repair of DNA replication errors. In Escherichia coli, initiation of MMR involves recognition of the mismatch by MutS, recruitment of MutL, activation of endonuclease MutH and DNA strand incision at a hemimethylated GATC site. Here, we studied the mechanism of communication that couples mismatch recognition to daughter strand incision. We investigated the effect of catalytically-deficient Cas9 as well as stalled RNA polymerase as roadblocks placed on DNA in between the mismatch and GATC site in ensemble and single molecule nanomanipulation incision assays. The MMR proteins were observed to incise GATC sites beyond a roadblock, albeit with reduced efficiency. This residual incision is completely abolished upon shortening the disordered linker regions of MutL. These results indicate that roadblock bypass can be fully attributed to the long, disordered linker regions in MutL and establish that communication during MMR initiation occurs along the DNA backbone., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

A shared paradigm of mismatch repair (MMR) across biology depicts extensive exonuclease-driven strand-specific excision that begins at a distant single-stranded DNA (ssDNA) break and proceeds back past the mismatched nucleotides. Historical reconstitution studies concluded that Escherichia coli (Ec) MMR employed EcMutS, EcMutL, EcMutH, EcUvrD, EcSSB and one of four ssDNA exonucleases to accomplish excision. Recent single-molecule images demonstrated that EcMutS and EcMutL formed cascading sliding clamps on a mismatched DNA that together assisted EcMutH in introducing ssDNA breaks at distant newly replicated GATC sites. Here we visualize the complete strand-specific excision process and find that long-lived EcMutL sliding clamps capture EcUvrD helicase near the ssDNA break, significantly increasing its unwinding processivity. EcSSB modulates the EcMutL-EcUvrD unwinding dynamics, which is rarely accompanied by extensive ssDNA exonuclease digestion. Together these observations are consistent with an exonuclease-independent MMR strand excision mechanism that relies on EcMutL-EcUvrD helicase-driven displacement of ssDNA segments between adjacent EcMutH-GATC incisions.

Meiotic recombination is essential for producing healthy gametes, and also generates genetic diversity. DNA double-strand break (DSB) formation is the initiating step of meiotic recombination, producing, among other outcomes, crossovers between homologous chromosomes (homologs), which provide physical links to guide accurate chromosome segregation. The parameters influencing DSB position and repair are thus crucial determinants of reproductive success and genetic diversity. Using Schizosaccharomyces pombe, we show that the distance between sequence polymorphisms across homologs has a strong impact on meiotic recombination rate. The closer the sequence polymorphisms are to each other across the homologs the fewer recombination events were observed. In the immediate vicinity of DSBs, sequence polymorphisms affect the frequency of intragenic recombination events (gene conversions). Additionally, and unexpectedly, the crossover rate of flanking markers tens of kilobases away from the sequence polymorphisms was affected by their relative position to each other amongst the progeny having undergone intragenic recombination. A major regulator of this distance-dependent effect is the MutSα-MutLα complex consisting of Msh2, Msh6, Mlh1, and Pms1. Additionally, the DNA helicases Rqh1 and Fml1 shape recombination frequency, although the effects seen here are largely independent of the relative position of the sequence polymorphisms.

Defective mismatch repair leads to increased mutation rates, and germline loss-of-function variants in the repair component MLH1 cause the hereditary cancer predisposition disorder known as Lynch syndrome. Early diagnosis is important, but complicated by many variants being of unknown significance. Here we show that a majority of the disease-linked MLH1 variants we studied are present at reduced cellular levels. We show that destabilized MLH1 variants are targeted for chaperone-assisted proteasomal degradation, resulting also in degradation of co-factors PMS1 and PMS2. In silico saturation mutagenesis and computational predictions of thermodynamic stability of MLH1 missense variants revealed a correlation between structural destabilization, reduced steady-state levels and loss-of-function. Thus, we suggest that loss of stability and cellular degradation is an important mechanism underlying many MLH1 variants in Lynch syndrome. Combined with analyses of conservation, the thermodynamic stability predictions separate disease-linked from benign MLH1 variants, and therefore hold potential for Lynch syndrome diagnostics., Competing Interests: AA, AS, SN, KS, EP, AS, EH, IB, AG, MT, CI, KL, RH No competing interests declared, (© 2019, Abildgaard et al.)

In Saccharomyces cerevisiae , the meiosis-specific axis proteins Hop1 and Red1 are present nonuniformly across the genome. In a previous study, the meiosis-specific VMA1-derived endonuclease (VDE) was used to examine Spo11-independent recombination in a recombination reporter inserted in a Hop1/Red1-enriched region ( HIS4 ) and in a Hop1/Red1-poor region ( URA3 ). VDE-initiated crossovers at HIS 4 were mostly dependent on Mlh3, a component of the MutLγ meiotic recombination intermediate resolvase, while VDE-initiated crossovers at URA3 were mostly Mlh3-independent. These differences were abolished in the absence of the chromosome axis remodeler Pch2, and crossovers at both loci became partly Mlh3-dependent. To test the generality of these observations, we examined inserts at six additional loci that differed in terms of Hop1/Red1 enrichment, chromosome size, and distance from centromeres and telomeres. All six loci behaved similarly to URA3 : the vast majority of VDE-initiated crossovers were Mlh3-independent. This indicates that, counter to previous suggestions, levels of meiotic chromosome axis protein enrichment alone do not determine which recombination pathway gives rise to crossovers during VDE-initiated meiotic recombination. In pch2∆ mutants, the fraction of VDE-induced crossovers that were Mlh3-dependent increased to levels previously observed for Spo11-initiated crossovers in pch2∆ , indicating that Pch2-dependent processes play an important role in controlling the balance between MutLγ-dependent and MutLγ-independent crossovers., (Copyright © 2019 Shodhan et al.)

MutL homolog 1 (MLH1) is a key DNA mismatch repair protein, which plays an important role in maintenance of genomic stability and the DNA damage response. Here, we report that MLH1 is a novel substrate of histone deacetylase 6 (HDAC6). HDAC6 interacts with and deacetylates MLH1 both in vitro and in vivo Interestingly, deacetylation of MLH1 blocks the assembly of the MutSα-MutLα complex. Moreover, we have identified four novel acetylation sites in MLH1 by MS analysis. The deacetylation mimetic mutant, but not the WT and the acetylation mimetic mutant, of MLH1 confers resistance to 6-thioguanine. Overall, our findings suggest that the MutSα-MutLα complex serves as a sensor for DNA damage response and that HDAC6 disrupts the MutSα-MutLα complex by deacetylation of MLH1, leading to the tolerance of DNA damage., (© 2019 Zhang et al.)

In eukaryotes and most bacteria, the MutS1/MutL-dependent mismatch repair system (MMR) corrects DNA mismatches that arise as replication errors. MutS1 recognizes mismatched DNA and stimulates the nicking endonuclease activity of MutL to incise mismatch-containing DNA. In archaea, there has been no experimental evidence to support the existence of the MutS1/MutL-dependent MMR. Instead, it was revealed that a large part of archaea possess mismatch-specific endonuclease EndoMS, indicating that the EndoMS-dependent MMR is widely adopted in archaea. However, some archaeal genomes encode MutS1 and MutL homologs, and their molecular functions have not been revealed. In this study, we purified and characterized recombinant MutS1 and the C-terminal endonuclease domain of MutL from a methanogenic archaeon Methanosaeta thermophila (mtMutS1 and the mtMutL CTD, respectively). mtMutS1 bound to mismatched DNAs with a higher affinity than to perfectly-matched and other structured DNAs, which resembles the DNA-binding specificities of eukaryotic and bacterial MutS1 homologs. The mtMutL CTD showed a Mn 2+ /Ni 2+ /Co 2+ -dependent nicking endonuclease activity that introduces single-strand breaks into a circular double-stranded DNA. The nicking endonuclease activity of the mtMutL CTD was impaired by mutagenizing the metal-binding motif that is identical to those of eukaryotic and bacterial MutL endonucleases. These results raise the possibility that not only the EndoMS-dependent MMR but also the traditional MutS1/MutL-dependent MMR exist in archaea., (Copyright © 2019 Elsevier B.V. All rights reserved.)

DNA mismatch repair is an evolutionarily conserved repair pathway that corrects replication errors. In most prokaryotes and all eukaryotes, the mismatch repair protein MutL is a sequence-unspecific endonuclease that nicks the newly synthesized strand and marks it for repair. Although the sequence of the endonuclease domain of MutL is not conserved, eukaryotic MutLα and prokaryotic MutL share four conserved motifs that define the endonuclease site of the protein. Their endonuclease activity is stimulated by the processivity sliding β-clamp, or its eukaryotic counterpart PCNA, highlighting the functional conservation. Bacterial MutL homologs form homodimers and, therefore, they have two endonuclease sites. However, eukaryotic MutL homologs associate to form heterodimers, where only one of the protomers of the dimer has endonuclease activity. To probe whether bacterial MutL needs its two endonuclease sites, we engineered variants of B. subtilis MutL harboring a single nuclease site and showed that these variants are functional nucleases. We also find that the protomer harboring the nuclease site must be able to bind to the β-clamp to recapitulate the nicking activity of wild-type MutL. These results demonstrate the functional asymmetry of bacterial MutL and strengthen the similarities with the endonuclease activity of eukaryotic MutL homologs., (Copyright © 2018 Elsevier B.V. All rights reserved.)

MutLα, a heterodimer consisting of MLH1 and PMS2, is a key player of DNA mismatch repair (MMR), yet little is known about its regulation. In this study, we used mass spectrometry to identify phosphorylated residues within MLH1 and PMS2. The most frequently detected phosphorylated amino acid was serine 477 of MLH1. Pharmacological treatment indicates‎ that Casein kinase II (CK2) could be responsible for the phosphorylation of MLH1 at serine 477 in vivo. In vitro kinase assay verified MLH1 as a substrate of CK2. Most importantly, using in vitro MMR assay we could demonstrate that p-MLH1 S477 lost MMR activity. Moreover, we found that levels of p-MLH1 S477 varied during the cell cycle. In summary, we identified that phosphorylation of MLH1 by CK2 at amino acid position 477 can switch off MMR activity in vitro. Since CK2 is overexpressed in many tumors and is able to inactivate MMR, the new mechanism here described could have an important impact on tumors overactive in CK2., (© 2018 Wiley Periodicals, Inc.)

DNA mismatch repair (MMR) is a DNA excision-resynthesis process that principally enhances replication fidelity. Highly conserved MutS (MSH) and MutL (MLH/PMS) homologs initiate MMR and in higher eukaryotes act as DNA damage sensors that can trigger apoptosis. MSH proteins recognize mismatched nucleotides, whereas the MLH/PMS proteins mediate multiple interactions associated with downstream MMR events including strand discrimination and strand-specific excision that are initiated at a significant distance from the mismatch. Remarkably, the biophysical functions of the MLH/PMS proteins have been elusive for decades. Here we consider recent observations that have helped to define the mechanics of MLH/PMS proteins and their role in choreographing MMR. We highlight the stochastic nature of DNA interactions that have been visualized by single-molecule analysis and the plasticity of protein complexes that employ thermal diffusion to complete the progressions of MMR., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Escherichia coli UvrD is a superfamily 1 helicase/translocase involved in multiple DNA metabolic processes including methyl-directed mismatch DNA repair. Although a UvrD monomer can translocate along single-stranded DNA, a UvrD dimer is needed for processive helicase activity in vitro. E. coli MutL, a regulatory protein involved in methyl-directed mismatch repair, stimulates UvrD helicase activity; however, the mechanism is not well understood. Using single-molecule fluorescence and ensemble approaches, we find that a single MutL dimer can activate latent UvrD monomer helicase activity. However, we also find that MutL stimulates UvrD dimer helicase activity. We further find that MutL enhances the DNA-unwinding processivity of UvrD. Hence, MutL acts as a processivity factor by binding to and presumably moving along with UvrD to facilitate DNA unwinding., (Copyright © 2018. Published by Elsevier Ltd.)

The Fragile X-related disorders (FXDs) are Repeat Expansion Diseases resulting from an expansion of a CGG-repeat tract at the 5' end of the FMR1 gene. The mechanism responsible for this unusual mutation is not fully understood. We have previously shown that mismatch repair (MMR) complexes, MSH2/MSH3 (MutSβ) and MSH2/MSH6 (MutSα), together with Polβ, a DNA polymerase important for base excision repair (BER), are important for expansions in a mouse model of these disorders. Here we show that MLH1/MLH3 (MutLγ), a protein complex that can act downstream of MutSβ in MMR, is also required for all germ line and somatic expansions. However, exonuclease I (EXO1), which acts downstream of MutL proteins in MMR, is not required. In fact, a null mutation in Exo1 results in more extensive germ line and somatic expansions than is seen in Exo1+/+ animals. Furthermore, mice homozygous for a point mutation (D173A) in Exo1 that eliminates its nuclease activity but retains its native conformation, shows a level of expansion that is intermediate between Exo1+/+ and Exo1-/- animals. Thus, our data suggests that expansion of the FX repeat in this mouse model occurs via a MutLγ-dependent, EXO1-independent pathway, with EXO1 protecting against expansion both in a nuclease-dependent and a nuclease-independent manner. Our data thus have implications for the expansion mechanism and add to our understanding of the genetic factors that may be modifiers of expansion risk in humans., Competing Interests: The authors have declared that no competing interests exist.