DNA repair proteins can be recruited by their histone reader domains to specific epigenomic features, with consequences on intragenomic mutation rate variation. Here, we investigated H3K4me1-associated hypomutation in plants. We first examined 2 proteins which, in plants, contain Tudor histone reader domains: PRECOCIOUS DISSOCIATION OF SISTERS 5 (PDS5C), involved in homology-directed repair, and MUTS HOMOLOG 6 (MSH6), a mismatch repair protein. The MSH6 Tudor domain of Arabidopsis (Arabidopsis thaliana) binds to H3K4me1 as previously demonstrated for PDS5C, which localizes to H3K4me1-rich gene bodies and essential genes. Mutations revealed by ultradeep sequencing of wild-type and msh6 knockout lines in Arabidopsis show that functional MSH6 is critical for the reduced rate of single-base substitution (SBS) mutations in gene bodies and H3K4me1-rich regions. We explored the breadth of these mechanisms among plants by examining a large rice (Oryza sativa) mutation data set. H3K4me1-associated hypomutation is conserved in rice as are the H3K4me1-binding residues of MSH6 and PDS5C Tudor domains. Recruitment of DNA repair proteins by H3K4me1 in plants reveals convergent, but distinct, epigenome-recruited DNA repair mechanisms from those well described in humans. The emergent model of H3K4me1-recruited repair in plants is consistent with evolutionary theory regarding mutation modifier systems and offers mechanistic insight into intragenomic mutation rate variation in plants.

DNA methylation is closely related to cancer. It is generally accepted that DNA methylation detection is crucial in cancer diagnosis, prognosis, and treatment monitoring. Therefore, there is an urgent demand for developing a simple, rapid, highly sensitive, and highly specific methylation detection method to detect DNA methylation at specific sites quantitatively. In this work, we introduce a DNA methylation detection method based on MutS and methylation-specific PCR, named MutS-based methylation-specific PCR (MB-MSP), which has the advantages of simplicity, speed, high specificity, sensitivity, and broad applicability. Utilizing the MutS\'s ability to bind mismatched base pairs, we inhibit not only the amplification of unmethylated DNA but also nonspecific primer amplification. We achieved a detection sensitivity of 0.5% for the methylated genes of ACP1, CLEC11A, and SEPT9 by MB-MSP. It has a good linear relationship and a detection time of only 1.5 h. To validate the feasibility of the MB-MSP method in clinical application, we conducted methylation detection on plasma-circulating tumor DNA samples from 10 liver cancer patients and 5 healthy people, achieving a 100% accuracy rate. In conclusion, MB-MSP, as a novel and reliable DNA methylation detection tool, holds significant application value and potential for advancing early cancer diagnosis.

MutS homolog 1 (MSH1) is involved in the recombining and repairing of organelle genomes and is essential for maintaining their stability. Previous studies indicated that the length of the gene varied greatly among species and detected species-specific partial gene duplications in Physcomitrella patens. However, there are critical gaps in the understanding of the gene size expansion, and the extent of the partial gene duplication of MSH1 remains unclear. Here, we screened MSH1 genes in 85 selected species with genome sequences representing the main clades of green plants (Viridiplantae). We identified the MSH1 gene in all lineages of green plants, except for nine incomplete species, for bioinformatics analysis. The gene is a singleton gene in most of the selected species with conserved amino acids and protein domains. Gene length varies greatly among the species, ranging from 3234 bp in Ostreococcus tauri to 805,861 bp in Cycas panzhihuaensis. The expansion of MSH1 repeatedly occurred in multiple clades, especially in Gymnosperms, Orchidaceae, and Chloranthus spicatus. MSH1 has exceptionally long introns in certain species due to the gene length expansion, and the longest intron even reaches 101,025 bp. And the gene length is positively correlated with the proportion of the transposable elements (TEs) in the introns. In addition, gene structure analysis indicated that the MSH1 of green plants had undergone parallel intron gains and losses in all major lineages. However, the intron number of seed plants (gymnosperm and angiosperm) is relatively stable. All the selected gymnosperms contain 22 introns except for Gnetum montanum and Welwitschia mirabilis, while all the selected angiosperm species preserve 21 introns except for the ANA grade. Notably, the coding region of MSH1 in algae presents an exceptionally high GC content (47.7% to 75.5%). Moreover, over one-third of the selected species contain species-specific partial gene duplications of MSH1, except for the conserved mosses-specific partial gene duplication. Additionally, we found conserved alternatively spliced MSH1 transcripts in five species. The study of MSH1 sheds light on the evolution of the long genes of green plants.

Lynch syndrome (LS) is an autosomal dominant inherited disorder, characterized by a predisposition to various cancers, mainly colorectal cancer (CRC). LS is caused by germline mutations in DNA mismatch repair genes i.e. mutL homolog 1 (MLH1), mutS homolog 2 (MSH2), mutS homolog 6 (MSH6), and post-meiotic segregation increased 2 (PMS2). In this study, we report a novel germline frameshift mutation in the MLH1 gene [NM_000249: exon1: c.99dup p.(Glu34ArgfsTer4)] in a 34-year-old male patient with LS. This MLH1 alteration has never been reported in any database or any publications. The frameshift mutation in MLH1 gene [NM_000249: exon1: c.99dup p.(Glu34ArgfsTer4)] was confirmed by Sanger sequencing conducted on peripheral blood of the proband. Meanwhile, Sanger sequencing results revealed the proband\'s uncle was the carrier. As multiple downstream germline frameshift mutations of this variation are pathogenic, such as MLH1 M35fs, N38fs, and S44fs, it is predicted that MLH1 p.(Glu34ArgfsTer4) might be also pathogenic. Meanwhile, this MLH1 mutation p.(Glu34ArgfsTer4) is predicted to be disease-causing by the MutationTaster software, as the duplication c.99dupA introduced a premature stop codon early in the translation, resulting in a non-functional protein. This study may contribute to the mutational spectrum of MLH1 leading to LS.

MutS homologs (MSHs) are highly conserved core components of DNA mismatch repair. Mismatch recognition provokes ATP-binding by MSH proteins that drives a conformational transition from a short-lived lesion-searching clamp to an extremely stable sliding clamp on the DNA. Here, we have expanded on previous bulk biochemical studies to examine the stability, lifetime, and kinetics of bacterial and human MSH sliding clamps on mismatched DNA using surface plasmon resonance and single-molecule analysis of fluorescently labeled proteins. We found that ATP-bound MSH complexes bound to blocked-end or very long mismatched DNAs were extremely stable over a range of ionic conditions. These observations underpinned the development of a high-throughput Förster resonance energy transfer system that specifically detects the formation of MSH sliding clamps on mismatched DNA. The Förster resonance energy transfer system is capable of distinguishing between HsMSH2-HsMSH3 and HsMSH2-HsMSH6 and appears suitable for chemical inhibitor screens. Taken together, our results provide additional insight into MSH sliding clamps as well as methods to distinguish their functions in mismatch repair.

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.

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.

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.

Trinucleotide repeats are a peculiar class of microsatellites whose expansions are responsible for approximately 30 human neurological or developmental disorders. The molecular mechanisms responsible for these expansions in humans are not totally understood, but experiments in model systems such as yeast, transgenic mice, and human cells have brought evidence that the mismatch repair machinery is involved in generating these expansions. The present review summarizes, in the first part, the role of mismatch repair in detecting and fixing the DNA strand slippage occurring during microsatellite replication. In the second part, key molecular differences between normal microsatellites and those that show a bias toward expansions are extensively presented. The effect of mismatch repair mutants on microsatellite expansions is detailed in model systems, and in vitro experiments on mismatched DNA substrates are described. Finally, a model presenting the possible roles of the mismatch repair machinery in microsatellite expansions is proposed.

A new biosensing method is presented to detect gene mutation by integrating the MutS protein from bacteria with a fiber optic particle plasmon resonance (FOPPR) sensing system. In this method, the MutS protein is conjugated with gold nanoparticles (AuNPs) deposited on an optical fiber core surface. The target double-stranded DNA containing an A and C mismatched base pair in a sample can be captured by the MutS protein, causing increased absorption of green light launching into the fiber and hence a decrease in transmitted light intensity through the fiber. As the signal change is enhanced through consecutive total internal reflections along the fiber, the limit of detection for an AC mismatch heteroduplex DNA can be as low as 0.49 nM. Because a microfluidic chip is used to contain the optical fiber, the narrow channel width allows an analysis time as short as 15 min. Furthermore, the label-free and real-time nature of the FOPPR sensing system enables determination of binding affinity and kinetics between MutS and single-base mismatched DNA. The method has been validated using a heterozygous PCR sample from a patient to determine the allelic fraction. The obtained allelic fraction of 0.474 reasonably agrees with the expected allelic fraction of 0.5. Therefore, the MutS-functionalized FOPPR sensor may potentially provide a convenient quantitative tool to detect single nucleotide polymorphisms in biological samples with a short analysis time at the point-of-care sites.