Interplay between H3K36me3, methyltransferase SETD2, and mismatch recognition protein MutSα facilitates processing of oxidative DNA damage in human cells.

Oxidative DNA damage contributes to aging and the pathogenesis of numerous human diseases including cancer. 8-hydroxyguanine (8-oxoG) is the major product of oxidative DNA lesions. Although OGG1-mediated base excision repair is the primary mechanism for 8-oxoG removal, DNA mismatch repair has also been implicated in processing oxidative DNA damage. However, the mechanism of the latter is not fully understood. Here, we treated human cells defective in various 8-oxoG repair factors with H2O2 and performed biochemical, live cell imaging, and chromatin immunoprecipitation sequencing analyses to determine their response to the treatment. We show that the mismatch repair processing of oxidative DNA damage involves cohesive interactions between mismatch recognition protein MutSα, histone mark H3K36me3, and H3K36 trimethyltransferase SETD2, which activates the ATM DNA damage signaling pathway. We found that cells depleted of MutSα or SETD2 accumulate 8-oxoG adducts and fail to trigger H2O2-induced ATM activation. Furthermore, we show that SETD2 physically interacts with both MutSα and ATM, which suggests a role for SETD2 in transducing DNA damage signals from lesion-bound MutSα to ATM. Consistently, MutSα and SETD2 are highly coenriched at oxidative damage sites. The data presented here support a model wherein MutSα, SETD2, ATM, and H3K36me3 constitute a positive feedback loop to help cells cope with oxidative DNA damage.

Complex mutation profiles in mismatch repair and ribonucleotide reductase mutants reveal novel repair substrate specificity of MutS homolog (MSH) complexes.

Determining mutation signatures is standard for understanding the etiology of human tumors and informing cancer treatment. Multiple determinants of DNA replication fidelity prevent mutagenesis that leads to carcinogenesis, including the regulation of free deoxyribonucleoside triphosphate pools by ribonucleotide reductase and repair of replication errors by the mismatch repair system. We identified genetic interactions between rnr1 alleles that skew and/or elevate deoxyribonucleoside triphosphate levels and mismatch repair gene deletions. These defects indicate that the rnr1 alleles lead to increased mutation loads that are normally acted upon by mismatch repair. We then utilized a targeted deep-sequencing approach to determine mutational profiles associated with mismatch repair pathway defects. By combining rnr1 and msh mutations to alter and/or increase deoxyribonucleoside triphosphate levels and alter the mutational load, we uncovered previously unreported specificities of Msh2-Msh3 and Msh2-Msh6. Msh2-Msh3 is uniquely able to direct the repair of G/C single-base deletions in GC runs, while Msh2-Msh6 specifically directs the repair of substitutions that occur at G/C dinucleotides. We also identified broader sequence contexts that influence variant profiles in different genetic backgrounds. Finally, we observed that the mutation profiles in double mutants were not necessarily an additive relationship of mutation profiles in single mutants. Our results have implications for interpreting mutation signatures from human tumors, particularly when mismatch repair is defective.

A Homozygous Loss-of-Function Mutation in MSH5 Abolishes MutSγ Axial Loading and Causes Meiotic Arrest in NOA-Affected Individuals.

Non-obstructive azoospermia (NOA), characterized by spermatogenesis failure and the absence of sperm in ejaculation, is the most severe form of male infertility. However, the etiology and pathology between meiosis-associated monogenic alterations and human NOA remain largely unknown. A homozygous MSH5 mutation (c.1126del) was identified from two idiopathic NOA patients in the consanguineous family. This mutation led to the degradation of MSH5 mRNA and abolished chromosome axial localization of MutSγ in spermatocytes from the affected males. Chromosomal spreading analysis of the patient's meiotic prophase I revealed that the meiosis progression was arrested at a zygotene-like stage with extensive failure of homologous synapsis and DSB repair. Therefore, our study demonstrates that the MSH5 c.1126del could cause meiotic recombination failure and lead to human infertility, improving the genetic diagnosis of NOA clinically. Furthermore, the study of human spermatocytes elucidates the meiosis defects caused by MSH5 variant, and reveals a conserved and indispensable role of MutSγ in human synapsis and meiotic recombination, which have not previously been well-described.

Clinicopathological, molecular features and prognosis of colorectal cancer with mucinous component.

Background: Colorectal cancer (CRC) with mucinous component is associated with distinct characteristics and controversial prognosis. Patients & methods: A total of 1800 CRC patients were retrospectively enrolled and grouped by the mucinous content of the primary tumors. The clinicopathological characteristics and overall survival rate were compared between groups. Results: Mucinous adenocarcinoma (MAC) and adenocarcinoma with mucinous component (AMC) had higher frequencies of DNA mismatch repair protein deficiency, KRAS, BRAF and PIK3CA mutations as compared to those of conventional adenocarcinoma (CAC). MAC had worse prognosis than CAC. However, MAC was not an independent prognostic factor in multivariable analysis. Conclusion: Molecular features of AMC and MAC were similar, which were different from those of CAC. Neither MAC nor AMC were independent prognostic factors for CRC.

MutSß Stimulates Holliday Junction Resolution by the SMX Complex.

MutSα and MutSß play important roles in DNA mismatch repair and are linked to inheritable cancers and degenerative disorders. Here, we show that MSH2 and MSH3, the two components of MutSß, bind SLX4 protein, a scaffold for the assembly of the SLX1-SLX4-MUS81-EME1-XPF-ERCC1 (SMX) trinuclease complex. SMX promotes the resolution of Holliday junctions (HJs), which are intermediates in homologous recombinational repair. We find that MutSß binds HJs and stimulates their resolution by SLX1-SLX4 or SMX in reactions dependent upon direct interactions between MutSß and SLX4. In contrast, MutSα does not stimulate HJ resolution. MSH3-depleted cells exhibit reduced sister chromatid exchanges and elevated levels of homologous recombination ultrafine bridges (HR-UFBs) at mitosis, consistent with defects in the processing of recombination intermediates. These results demonstrate a role for MutSß in addition to its established role in the pathogenic expansion of CAG/CTG trinucleotide repeats, which is causative of myotonic dystrophy and Huntington's disease.

PCNA activates the MutLγ endonuclease to promote meiotic crossing over.

During meiosis, crossover recombination connects homologous chromosomes to direct their accurate segregation1. 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 junctions2,3. A central principle of crossover resolution is that the two Holliday junctions are resolved in opposite planes by targeting nuclease incisions to specific DNA strands4. The endonuclease activity of the MutLγ complex has been implicated in the resolution of crossovers5-10, 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 junctions11. 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 repair12,13 and evoke a novel model for crossover-specific resolution of double Holliday junctions during meiosis.

Regulation of the MLH1-MLH3 endonuclease in meiosis.

During prophase of the first meiotic division, cells deliberately break their DNA1. These DNA breaks are repaired by homologous recombination, which facilitates proper chromosome segregation and enables the reciprocal exchange of DNA segments between homologous chromosomes2. 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 mechanism3-7. Here we have biochemically reconstituted key elements of this pro-crossover pathway. We show that human MSH4-MSH5 (MutSγ), which supports crossing over8, 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 points9. 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 chromosomes4.

Chromatin remodeling and mismatch repair: Access and excision.

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.

Interactome Analysis and Docking Sites of MutS Homologs Reveal New Physiological Roles in Arabidopsis thaliana.

Due to their sedentary lifestyle, plants are constantly exposed to different stress stimuli. Stress comes in variety of forms where factors like radiation, free radicals, "replication errors, polymerase slippage", and chemical mutagens result in genotoxic or cytotoxic damage. In order to face "the base oxidation or DNA replication stress", plants have developed many sophisticated mechanisms. One of them is the DNA mismatch repair (MMR) pathway. The main part of the MMR is the MutS homologue (MSH) protein family. The genome of Arabidopsis thaliana encodes at least seven homologues of the MSH family: AtMSH1, AtMSH2, AtMSH3, AtMSH4, AtMSH5, AtMSH6, and AtMSH7. Despite their importance, the functions of AtMSH homologs have not been investigated. In this work, bioinformatics tools were used to obtain a better understanding of MSH-mediated DNA repair mechanisms in Arabidopsis thaliana and to understand the additional biological roles of AtMSH family members. In silico analysis, including phylogeny tracking, prediction of 3D structure, interactome analysis, and docking site prediction, suggested interactions with proteins were important for physiological development of A. thaliana. The MSH homologs extensively interacted with both TIL1 and TIL2 (DNA polymerase epsilon catalytic subunit), proteins involved in cell fate determination during plant embryogenesis and involved in flowering time repression. Additionally, interactions with the RECQ protein family (helicase enzymes) and proteins of nucleotide excision repair pathway were detected. Taken together, the results presented here confirm the important role of AtMSH proteins in mismatch repair and suggest important new physiological roles.

Activity of Vsr endonucleases encoded by Neisseria gonorrhoeae FA1090 is influenced by MutL and MutS proteins.

BACKGROUND: The functioning of DNA repair systems is based on correct interactions between proteins involved in DNA repair. Very Short Patch (VSP) repair is a DNA repair system that corrects mismatches resulting from the deamination of 5-methylcytosine. The key enzyme in the VSP system is Vsr endonuclease, which can cleave mismatched DNA independently of accessory proteins. Until now, in vivo activity has only been shown for V.EcoKDcm - the only Vsr endonuclease in Escherichia coli. Additionally, the VSP system of E. coli is the only one for which interactions between proteins of the system have been demonstrated. Neisseria gonorrhoeae FA1090 is the first bacterium that we previously demonstrated to encode two active in vitro Vsr endonucleases: V.NgoAXIII and V.NgoAXIV. RESULTS: We elucidate the mutator phenotype of N. gonorrhoeae mutants with disrupted genes encoding V.NgoAXIII or V.NgoAXIV endonuclease. Furthermore, we investigate the interactions between gonococcal Vsr endonucleases and MutL and MutS proteins. The Vsr endonucleases physically interact with gonococcal MutL protein but not with MutS protein. In the presence of the MutL protein, the efficiency of DNA cleavage by both V.NgoAXIII and V.NgoAXIV endonucleases increases, resulting in a decrease in the amount of Vsr enzyme required to complete digestion of mismatched DNA. Both Vsr endonucleases are also stimulated in vitro by the MutL protein of E. coli. In turn, the gonococcal MutS protein hinders DNA cleavage by the Vsr endonucleases. However, this effect is overridden in the presence of MutL, and furthermore, the simultaneous presence of MutL and MutS causes an increase in the efficiency of DNA cleavage by the Vsr endonucleases compared to the reaction catalyzed by V.NgoAXIII or V.NgoAXIV alone. CONCLUSIONS: For the first time, interactions between proteins of the DNA repair system encoded by N. gonorrhoeae that are responsible for the correction of mismatches resulting from the 5-methylcytosine deamination were identified. The increase in activity of Vsr endonucleases in the presence of MutL protein could allow for reduced synthesis of the Vsr endonucleases in cells, and the susceptibility of gonococcal Vsr endonucleases on MutL protein of E. coli implies a universal mechanism of Vsr stimulation by MutL protein.

H3K36me3-mediated mismatch repair preferentially protects actively transcribed genes from mutation.

Histone H3 trimethylation at lysine 36 (H3K36me3) is an important histone mark involved in both transcription elongation and DNA mismatch repair (MMR). It is known that H3K36me3 recruits the mismatch-recognition protein MutSα to replicating chromatin via its physical interaction with MutSα's PWWP domain, but the exact role of H3K36me3 in transcription is undefined. Using ChIP combined with whole-genome DNA sequencing analysis, we demonstrate here that H3K36me3, together with MutSα, is involved in protecting against mutation, preferentially in actively transcribed genomic regions. We found that H3K36me3 and MutSα are much more co-enriched in exons and actively transcribed regions than in introns and nontranscribed regions. The H3K36me3-MutSα co-enrichment correlated with a much lower mutation frequency in exons and actively transcribed regions than in introns and nontranscribed regions. Correspondingly, depleting H3K36me3 or disrupting the H3K36me3-MutSα interaction elevated the spontaneous mutation frequency in actively transcribed genes, but it had little influence on the mutation frequency in nontranscribed or transcriptionally inactive regions. Similarly, H2O2-induced mutations, which mainly cause base oxidations, preferentially occurred in actively transcribed genes in MMR-deficient cells. The data presented here suggest that H3K36me3-mediated MMR preferentially safeguards actively transcribed genes not only during replication by efficiently correcting mispairs in early replicating chromatin but also during transcription by directly or indirectly removing DNA lesions associated with a persistently open chromatin structure.

Mismatch repair system proteins in oral benign and malignant lesions.

Different environmental agents may cause DNA mutations by disrupting its double-strand structure; however, even normal DNA polymerase function may synthesize mismatch nucleotide bases, occasionally demonstrating failure in its proofreading activity. To overcome this issue, mismatch repair (MMR) system, a group of proteins specialized in finding mispairing bases and small loops of insertion or deletion, works to avoid the occurrence of mutations that could ultimately lead to innumerous human diseases. In the last decades, the role of MMR proteins in oral carcinogenesis and in the development of other oral cavity neoplasms has grown, but their importance in the pathogenesis and their prognostic potential for patients affected by oral malignancies, especially oral squamous cell carcinoma (OSCC), remain unclear. Therefore, in this manuscript we aimed to review and critically discuss the currently available data on MMR proteins expression in oral potentially malignant lesions, in OSCC, and in other oral neoplasms to better understand their relevance in these lesions.

MutSß promotes trinucleotide repeat expansion by recruiting DNA polymerase ß to nascent (CAG)n or (CTG)n hairpins for error-prone DNA synthesis.

Expansion of (CAG)•(CTG) repeats causes a number of familial neurodegenerative disorders. Although the underlying mechanism remains largely unknown, components involved in DNA mismatch repair, particularly mismatch recognition protein MutSß (a MSH2-MSH3 heterodimer), are implicated in (CAG)•(CTG) repeat expansion. In addition to recognizing small insertion-deletion loop-outs, MutSß also specifically binds DNA hairpin imperfect heteroduplexes formed within (CAG)n•(CTG)n sequences. However, whether or not and how MutSß binding triggers expansion of (CAG)•(CTG) repeats remain unknown. We show here that purified recombinant MutSß physically interacts with DNA polymerase ß (Polß) and stimulates Polß-catalyzed (CAG)n or (CTG)n hairpin retention. Consistent with these in vitro observations, MutSß and Polß interact with each other in vivo, and colocalize at (CAG)•(CTG) repeats during DNA replication. Our data support a model for error-prone processing of (CAG)n or (CTG)n hairpins by MutSß and Polß during DNA replication and/or repair: MutSß recognizes (CAG)n or (CTG)n hairpins formed in the nascent DNA strand, and recruits Polß to the complex, which then utilizes the hairpin as a primer for extension, leading to (CAG)•(CTG) repeat expansion. This study provides a novel mechanism for trinucleotide repeat expansion in both dividing and non-dividing cells.