Spontaneous and double-strand break repair-associated quasipalindrome and frameshift mutagenesis in budding yeast: role of mismatch repair.

Although gene conversion (GC) in Saccharomyces cerevisiae is the most error-free way to repair double-strand breaks (DSBs), the mutation rate during homologous recombination is 1,000 times greater than during replication. Many mutations involve dissociating a partially copied strand from its repair template and re-aligning with the same or another template, leading to -1 frameshifts in homonucleotide runs, quasipalindrome (QP)-associated mutations and microhomology-mediated interchromosomal template switches. We studied GC induced by HO endonuclease cleavage at MATα, repaired by an HMR::KI-URA3 donor. We inserted into HMR::KI-URA3 an 18-bp inverted repeat where one arm had a 4-bp insertion. Most GCs yield MAT::KI-ura3::QP + 4 (Ura-) outcomes, but template-switching produces Ura+ colonies, losing the 4-bp insertion. If the QP arm without the insertion is first encountered by repair DNA polymerase and is then (mis)used as a template, the palindrome is perfected. When the QP + 4 arm is encountered first, Ura+ derivatives only occur after second-end capture and second-strand synthesis. QP + 4 mutations are suppressed by mismatch repair (MMR) proteins Msh2, Msh3, and Mlh1, but not Msh6. Deleting Rdh54 significantly reduces QP mutations only when events creating Ura+ occur in the context of a D-loop but not during second-strand synthesis. A similar bias is found with a proofreading-defective DNA polymerase mutation (poI3-01). DSB-induced mutations differed in several genetic requirements from spontaneous events. We also created a + 1 frameshift in the donor, expanding a run of 4 Cs to 5 Cs. Again, Ura+ recombinants markedly increased by disabling MMR, suggesting that MMR acts during GC but favors the unbroken, template strand.

Therapeutic validation of MMR-associated genetic modifiers in a human ex vivo model of Huntington disease.

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.

Microsatellite instability in mismatch repair proficient colorectal cancer: clinical features and underlying molecular mechanisms.

BACKGROUND: Both defects in mismatch repair (dMMR) and high microsatellite instability (MSI-H) have been recognised as crucial biomarkers that guide treatment strategies and disease management in colorectal cancer (CRC). As MMR and MSI tests are being widely conducted, an increasing number of MSI-H tumours have been identified in CRCs with mismatch repair proficiency (pMMR). The objective of this study was to assess the clinical features of patients with pMMR/MSI-H CRC and elucidate the underlying molecular mechanism in these cases. METHODS: From January 2015 to December 2018, 1684 cases of pMMR and 401 dMMR CRCs were enrolled. Of those patients, 93 pMMR/MSI-H were identified. The clinical phenotypes and prognosis were analysed. Frozen and paraffin-embedded tissue were available in 35 patients with pMMR/MSI-H, for which comprehensive genomic and transcriptomic analyses were performed. FINDINGS: In comparison to pMMR/MSS CRCs, pMMR/MSI-H CRCs exhibited significantly less tumour progression and better long-term prognosis. The pMMR/MSI-H cohorts displayed a higher presence of CD8+ T cells and NK cells when compared to the pMMR/MSS group. Mutational signature analysis revealed that nearly all samples exhibited deficiencies in MMR genes, and we also identified deleterious mutations in MSH3-K383fs. INTERPRETATION: This study revealed pMMR/MSI-H CRC as a distinct subgroup within CRC, which manifests diverse clinicopathological features and long-term prognostic outcomes. Distinct features in the tumour immune-microenvironment were observed in pMMR/MSI-H CRCs. Pathogenic deleterious mutations in MSH3-K383fs were frequently detected, suggesting another potential biomarker for identifying MSI-H. FUNDING: This work was supported by the Science and Technology Commission of Shanghai Municipality (20DZ1100101).

High-throughput sequencing and in-silico analysis confirm pathogenicity of novel MSH3 variants in African American colorectal cancer.

The maintenance of DNA sequence integrity is critical to avoid accumulation of cancer-causing mutations. Inactivation of DNA Mismatch Repair (MMR) genes (e.g., MLH1 and MSH2) is common among many cancers, including colorectal cancer (CRC) and is the driver of classic microsatellite instability (MSI) in tumors. Somatic MSH3 alterations have been linked to a specific form of MSI called elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) that is associated with patient poor prognosis and elevated among African American (AA) rectal cancer patients. Genetic variants of MSH3 and their pathogenicity vary among different populations, such as among AA, which are not well-represented in publicly available databases. Targeted exome sequencing of MSH3 among AA CRC samples followed by computational bioinformatic pipeline and molecular dynamic simulation analysis approach confirmed six identified MSH3 variants (c.G1237A, c.C2759T, c.G1397A, c.G2926A, c.C3028T, c.G3241A) that corresponded to MSH3 amino-acid changes (p.E413K; p.S466N; p.S920F; p.E976K; p.H1010Y; p.E1081K). All identified MSH3 variants were non-synonymous, novel, pathogenic, and show loss or gain of hydrogen bonding, ionic bonding, hydrophobic bonding, and disulfide bonding and have a deleterious effect on the structure of MSH3 protein. Some variants were located within the ATPase site of MSH3, affecting ATP hydrolysis that is critical for MSH3's function. Other variants were in the MSH3-MSH2 interacting domain, important for MSH3's binding to MSH2. Overall, our data suggest that these variants among AA CRC patients affect the function of MSH3 making them pathogenic and likely contributing to the development or advancement of CRC among AA. Further clarifying functional studies will be necessary to fully understand the impact of these variants on MSH3 function and CRC development in AA patients.

Elevated MSH2 MSH3 expression interferes with DNA metabolism in vivo.

The Msh2-Msh3 mismatch repair (MMR) complex in Saccharomyces cerevisiae recognizes and directs repair of insertion/deletion loops (IDLs) up to ∼17 nucleotides. Msh2-Msh3 also recognizes and binds distinct looped and branched DNA structures with varying affinities, thereby contributing to genome stability outside post-replicative MMR through homologous recombination, double-strand break repair (DSBR) and the DNA damage response. In contrast, Msh2-Msh3 promotes genome instability through trinucleotide repeat (TNR) expansions, presumably by binding structures that form from single-stranded (ss) TNR sequences. We previously demonstrated that Msh2-Msh3 binding to 5' ssDNA flap structures interfered with Rad27 (Fen1 in humans)-mediated Okazaki fragment maturation (OFM) in vitro. Here we demonstrate that elevated Msh2-Msh3 levels interfere with DNA replication and base excision repair in vivo. Elevated Msh2-Msh3 also induced a cell cycle arrest that was dependent on RAD9 and ELG1 and led to PCNA modification. These phenotypes also required Msh2-Msh3 ATPase activity and downstream MMR proteins, indicating an active mechanism that is not simply a result of Msh2-Msh3 DNA-binding activity. This study provides new mechanistic details regarding how excess Msh2-Msh3 can disrupt DNA replication and repair and highlights the role of Msh2-Msh3 protein abundance in Msh2-Msh3-mediated genomic instability.

Novel insights into the ecDNA formation mechanism involving MSH3 in methotrexate­resistant human colorectal cancer cells.

Extrachromosomal DNAs (ecDNAs), also known as double minutes (DMs), can induce a fast increase in gene copy numbers and promote the development of cancer, including drug resistance. MutS homolog 3 (MSH3), a key protein in mismatch repair, has been indicated to participate in the regulation of DNA double­strand break (DSB) repair, which has been reported to be associated with the formation of ecDNAs. However, it remains unclear whether MSH3 can influence drug resistance via ecDNAs in cancer. In the present study, high MSH3 expression was observed in methotrexate (MTX)­resistant HT29 cells [DM­ and homogeneously staining region (HSR)­containing cells] compared with parental HT29 cells. Additionally, decreased amounts of ecDNAs, HSRs and amplified genes locating on ecDNAs and HSRs were detected following depletion of MSH3 and this could be reversed by overexpressing MSH3 in DM­containing cells. No corresponding changes were found in HSR­containing cells. The present study further verified the involvement of MSH3­regulated DNA DSB repair pathways in the formation of ecDNAs by detecting the expression of core proteins and pathway activity. Furthermore, expulsion of ecDNAs/HSRs was detected and increased frequencies of micronuclei/nuclear buds with dihydrofolate reductase (DHFR) signals were observed in MSH3­depleted DM­containing cells. Finally, changes in MSH3 expression could affect DHFR amplification­derived DHFR expression and cell sensitivity to MTX, suggesting that MSH3 may influence cancer drug resistance by altering the amount of ecDNAs. In conclusion, the present study revealed a novel mechanism involving MSH3 in the regulation of ecDNAs by DSB repair, which will have clinical value in the treatment of ecDNA­based drug resistance in cancer.

MSH3: a confirmed predisposing gene for adenomatous polyposis.

BACKGROUND: The MSH3 gene is part of the DNA mismatch repair system, but has never been shown to be involved in Lynch syndrome. A first report of four patients from two families, bearing biallelic MSH3 germline variants, with a phenotype of attenuated colorectal adenomatous polyposis raised the question of its involvement in hereditary cancer predisposition. The patients' tumours exhibited elevated microsatellite alterations at selected tetranucleotide repeats (EMAST), a hallmark of MSH3 deficiency. METHODS: We report five new unrelated patients with MSH3-associated polyposis. We describe their personal and familial history and study the EMAST phenotype in various normal and tumour samples, which are relevant findings based on the rarity of this polyposis subtype so far. RESULTS: All patients had attenuated colorectal adenomatous polyposis, with duodenal polyposis in two cases. Both women had breast carcinomas. EMAST phenotype was present at various levels in different samples of the five patients, confirming the MSH3 deficiency, with a gradient of instability in polyps depending on their degree of dysplasia. The negative EMAST phenotype ruled out the diagnosis of germline MSH3 deficiency for two patients: one homozygous for a benign variant and one with a monoallelic large deletion. CONCLUSION: This report lends further credence to biallelic MSH3 germline pathogenic variants being involved in colorectal and duodenal adenomatous polyposis. Large-scale studies may help clarify the tumour spectrum and associated risks. Ascertainment of EMAST may help with the interpretation of variants of unknown significance. We recommend adding MSH3 to dedicated diagnostic gene panels.

MSH2-MSH3 promotes DNA end resection during homologous recombination and blocks polymerase theta-mediated end-joining through interaction with SMARCAD1 and EXO1.

DNA double-strand break (DSB) repair via homologous recombination is initiated by end resection. The extent of DNA end resection determines the choice of the DSB repair pathway. Nucleases for end resection have been extensively studied. However, it is still unclear how the potential DNA structures generated by the initial short resection by MRE11-RAD50-NBS1 are recognized and recruit proteins, such as EXO1, to DSB sites to facilitate long-range resection. We found that the MSH2-MSH3 mismatch repair complex is recruited to DSB sites through interaction with the chromatin remodeling protein SMARCAD1. MSH2-MSH3 facilitates the recruitment of EXO1 for long-range resection and enhances its enzymatic activity. MSH2-MSH3 also inhibits access of POLθ, which promotes polymerase theta-mediated end-joining (TMEJ). Collectively, we present a direct role of MSH2-MSH3 in the initial stages of DSB repair by promoting end resection and influencing the DSB repair pathway by favoring homologous recombination over TMEJ.

SYVN1-mediated ubiquitination and degradation of MSH3 promotes the apoptosis of lens epithelial cells.

The pathology of age-related cataract (ARC) mainly involves the misfolding and aggregation of proteins, especially oxidative damage repair proteins, in the lens, induced by ultraviolet-B (UVB). MSH3, as a key member of the mismatch repair family, primarily maintains genome stability. However, the function of MSH3 and the mechanism by which cells maintain MSH3 proteostasis during cataractogenesis remains unknown. In the present study, the protein expression levels of MSH3 were found to be attenuated in ARC specimens and SRA01/04 cells under UVB exposure. The ectopic expression of MSH3 notably impeded UVB-induced apoptosis, whereas the knockdown of MSH3 promoted apoptosis. Protein half-life assay revealed that UVB irradiation accelerated the decline of MSH3 by ubiquitination and degradation. Subsequently, we found that E3 ubiquitin ligase synoviolin (SYVN1) interacted with MSH3 and promoted its ubiquitination and degradation. Of note, the expression and function of SYVN1 were contrary to those of MSH3 and SYVN1 regulated MSH3 protein degradation via the ubiquitin-proteasome pathway and the autophagy-lysosome pathway. Based on these findings, we propose a mechanism for ARC pathogenesis that involves SYVN1-mediated degradation of MSH3 via the ubiquitin-proteasome pathway and the autophagy-lysosome pathway, and suggest that interventions targeting SYVN1 might be a potential therapeutic strategy for ARC.

FAN1's protection against CGG repeat expansion requires its nuclease activity and is FANCD2-independent.

The Repeat Expansion Diseases, a large group of human diseases that includes the fragile X-related disorders (FXDs) and Huntington's disease (HD), all result from expansion of a disease-specific microsatellite via a mechanism that is not fully understood. We have previously shown that mismatch repair (MMR) proteins are required for expansion in a mouse model of the FXDs, but that the FANCD2 and FANCI associated nuclease 1 (FAN1), a component of the Fanconi anemia (FA) DNA repair pathway, is protective. FAN1's nuclease activity has been reported to be dispensable for protection against expansion in an HD cell model. However, we show here that in a FXD mouse model a point mutation in the nuclease domain of FAN1 has the same effect on expansion as a null mutation. Furthermore, we show that FAN1 and another nuclease, EXO1, have an additive effect in protecting against MSH3-dependent expansions. Lastly, we show that the loss of FANCD2, a vital component of the Fanconi anemia DNA repair pathway, has no effect on expansions. Thus, FAN1 protects against MSH3-dependent expansions without diverting the expansion intermediates into the canonical FA pathway and this protection depends on FAN1 having an intact nuclease domain.

Profiling diverse sequence tandem repeats in colorectal cancer reveals co-occurrence of microsatellite and chromosomal instability involving Chromosome 8.

We developed a sensitive sequencing approach that simultaneously profiles microsatellite instability, chromosomal instability, and subclonal structure in cancer. We assessed diverse repeat motifs across 225 microsatellites on colorectal carcinomas. Our study identified elevated alterations at both selected tetranucleotide and conventional mononucleotide repeats. Many colorectal carcinomas had a mix of genomic instability states that are normally considered exclusive. An MSH3 mutation may have contributed to the mixed states. Increased copy number of chromosome arm 8q was most prevalent among tumors with microsatellite instability, including a case of translocation involving 8q. Subclonal analysis identified co-occurring driver mutations previously known to be exclusive.

FAN1 controls mismatch repair complex assembly via MLH1 retention to stabilize CAG repeat expansion in Huntington's disease.

CAG repeat expansion in the HTT gene drives Huntington's disease (HD) pathogenesis and is modulated by DNA damage repair pathways. In this context, the interaction between FAN1, a DNA-structure-specific nuclease, and MLH1, member of the DNA mismatch repair pathway (MMR), is not defined. Here, we identify a highly conserved SPYF motif at the N terminus of FAN1 that binds to MLH1. Our data support a model where FAN1 has two distinct functions to stabilize CAG repeats. On one hand, it binds MLH1 to restrict its recruitment by MSH3, thus inhibiting the assembly of a functional MMR complex that would otherwise promote CAG repeat expansion. On the other hand, it promotes accurate repair via its nuclease activity. These data highlight a potential avenue for HD therapeutics in attenuating somatic expansion.

Coordinated roles of SLX4 and MutSß in DNA repair and the maintenance of genome stability.

SLX4 provides a molecular scaffold for the assembly of multiple protein complexes required for the maintenance of genome stability. It is involved in the repair of DNA crosslinks, the resolution of recombination intermediates, the response to replication stress and the maintenance of telomere length. To carry out these diverse functions, SLX4 interacts with three structure-selective endonucleases, MUS81-EME1, SLX1 and XPF-ERCC1, as well as the telomere binding proteins TRF2, RTEL1 and SLX4IP. Recently, SLX4 was shown to interact with MutSß, a heterodimeric protein involved in DNA mismatch repair, trinucleotide repeat instability, crosslink repair and recombination. Importantly, MutSß promotes the pathogenic expansion of CAG/CTG trinucleotide repeats, which is causative of myotonic dystrophy and Huntington's disease. The colocalization and specific interaction of MutSß with SLX4, together with their apparently overlapping functions, are suggestive of a common role in reactions that promote DNA maintenance and genome stability. This review will focus on the role of SLX4 in DNA repair, the interplay between MutSß and SLX4, and detail how they cooperate to promote recombinational repair and DNA crosslink repair. Furthermore, we speculate that MutSß and SLX4 may provide an alternative cellular mechanism that modulates trinucleotide instability.

Prerecognition Diffusion Mechanism of Human DNA Mismatch Repair Proteins along DNA: Msh2-Msh3 versus Msh2-Msh6.

DNA mismatch repair (MMR) is an important postreplication process that eliminates mispaired or unpaired nucleotides to ensure genomic replication fidelity. In humans, Msh2-Msh6 and Msh2-Msh3 are the two mismatch repair initiation factors that recognize DNA lesions. While X-ray crystal structures exist for these proteins in complex with DNA lesions, little is known about their structures during the initial search along nonspecific double-stranded DNA, because they are short-lived and difficult to determine experimentally. In this study, various computational approaches were used to sidestep these difficulties. All-atom and coarse-grained simulations based on the crystal structures of Msh2-Msh3 and Msh2-Msh6 showed no translation along the DNA, suggesting that the initial search conformation differs from the lesion-bound crystal structure. We modeled probable search-mode structures of MSH proteins and showed, using coarse-grained molecular dynamics simulations, that they can perform rotation-coupled diffusion on DNA, which is a suitable and efficient search mechanism for their function and one predicted earlier by fluorescence resonance energy transfer and fluorescence microscopy studies. This search mechanism is implemented by electrostatic interactions among the mismatch-binding domain (MBD), the clamp domains, and the DNA backbone. During simulations, their diffusion rate did not change significantly with an increasing salt concentration, which is consistent with observations from experimental studies. When the gap between their DNA-binding clamps was increased, Msh2-Msh3 diffused mostly via the clamp domains while Msh2-Msh6 still diffused using the MBD, reproducing the experimentally measured lower diffusion coefficient of Msh2-Msh6. Interestingly, Msh2-Msh3 was capable of dissociating from the DNA, whereas Msh2-Msh6 always diffused on the DNA duplex. This is consistent with the experimental observation that Msh2-Msh3, unlike Msh2-Msh6, can overcome obstacles such as nucleosomes. Our models provide a molecular picture of the different mismatch search mechanisms undertaken by Msh2-Msh6 and Msh2-Msh3, despite the similarity of their structures.

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.

MutSα deficiency increases tolerance to DNA damage in yeast lacking postreplication repair.

By combining mutations in DNA repair genes, important and unexpected interactions between different repair pathways can be discovered. In this study, we identified a novel link between mismatch repair (MMR) genes and postreplication repair (PRR) in Saccharomyces cerevisiae. Strains lacking Rad5 (HLTF in mammals), a protein important for restarting stalled replication forks in the error-free PRR pathway, were supersensitive to the DNA methylating agent methyl methanesulfonate (MMS). Deletion of the mismatch repair genes, MSH2 or MSH6, which together constitutes the MutSα complex, partially suppressed the MMS super-sensitivity of the rad5Δ strain. Deletion of MSH2 also suppressed the MMS sensitivity of mms2Δ, which acts together with Rad5 in error-free PRR. However, inactivating the mismatch repair genes MSH3 and MLH1 did not suppress rad5Δ, showing that the suppression was specific for disabling MutSα. The partial suppression did not require translesion DNA synthesis (REV1, REV3 or RAD30), base excision repair (MAG1) or homologous recombination (RAD51). Instead, the underlying mechanism was dependent on RAD52 while independent of established pathways involving RAD52, like single-strand annealing and break-induced replication. We propose a Rad5- and Rad51-independent template switch pathway, capable of compensating for the loss of the error-free template-switch subpathway of postreplication repair, triggered by the loss of MutSα.

The Human DNA Mismatch Repair Protein MSH3 Contains Nuclear Localization and Export Signals That Enable Nuclear-Cytosolic Shuttling in Response to Inflammation.

Inactivation of DNA mismatch repair propels colorectal cancer (CRC) tumorigenesis. CRCs exhibiting elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) show reduced nuclear MutS homolog 3 (MSH3) expression with surrounding inflammation and portend poor patient outcomes. MSH3 reversibly exits from the nucleus to the cytosol in response to the proinflammatory cytokine interleukin-6 (IL-6), suggesting that MSH3 may be a shuttling protein. In this study, we manipulated three putative nuclear localization (NLS1 to -3) and two potential nuclear export signals (NES1 and -2) within MSH3. We found that both NLS1 and NLS2 possess nuclear import function, with NLS1 responsible for nuclear localization within full-length MSH3. We also found that NES1 and NES2 work synergistically to maximize nuclear export, with both being required for IL-6-induced MSH3 export. We examined a 27-bp deletion (Δ27bp) within the polymorphic exon 1 that occurs frequently in human CRC cells and neighbors NLS1. With oxidative stress, MSH3 with this deletion (Δ27bp MSH3) localizes to the cytoplasm, suggesting that NLS1 function in Δ27bp MSH3 is compromised. Overall, MSH3's shuttling in response to inflammation enables accumulation in the cytoplasm; reduced nuclear MSH3 increases EMAST and DNA damage. We suggest that polymorphic sequences adjacent to NLS1 may enhance cytosolic retention, which has clinical implications for inflammation-associated neoplastic processes.

Environmentally relevant exposure to dibutyl phthalate disrupts DNA damage repair gene expression in the mouse ovary†.

Phthalates have a history of reproductive toxicity in animal models and associations with adverse reproductive outcomes in women. Human exposure to dibutyl phthalate (DBP) occurs via consumer products (7-10 µg/kg/day) and medications (1-233 µg/kg/day). Most DBP toxicity studies have focused on high supraphysiological exposure levels; thus, very little is known about exposures occurring at environmentally relevant levels. CD-1 female mice (80 days old) were treated with tocopherol-stripped corn oil (vehicle control) or DBP dissolved in oil at environmentally relevant (10 and 100 µg/kg/day) or higher (1000 µg/kg/day) levels for 30 days to evaluate effects on DNA damage response (DDR) pathway genes and folliculogenesis. DBP exposure caused dose-dependent effects on folliculogenesis and gene expression. Specifically, animals exposed to the high dose of DBP had more atretic follicles in their ovaries, while in those treated with environmentally relevant doses, follicle numbers were no different from vehicle-treated controls. DBP exposure significantly reduced the expression of DDR genes including those involved in homologous recombination (Atm, Brca1, Mre11a, Rad50), mismatch repair (Msh3, Msh6), and nucleotide excision repair (Xpc, Pcna) in a dose-specific manner. Interestingly, staining for the DNA damage marker, γH2AX, was similar between treatments. DBP exposure did not result in differential DNA methylation in the Brca1 promoter but significantly reduced transcript levels for the maintenance DNA methyltransferase, Dnmt1, in the ovary. Collectively, these findings show that oral exposure to environmentally relevant levels of DBP for 30 days does not significantly impact folliculogenesis in adult mice but leads to aberrant ovarian expression of DDR genes.

Effective mismatch repair depends on timely control of PCNA retention on DNA by the Elg1 complex.

Proliferating cell nuclear antigen (PCNA) is a sliding clamp that acts as a central co-ordinator for mismatch repair (MMR) as well as DNA replication. Loss of Elg1, the major subunit of the PCNA unloader complex, causes over-accumulation of PCNA on DNA and also increases mutation rate, but it has been unclear if the two effects are linked. Here we show that timely removal of PCNA from DNA by the Elg1 complex is important to prevent mutations. Although premature unloading of PCNA generally increases mutation rate, the mutator phenotype of elg1Δ is attenuated by PCNA mutants PCNA-R14E and PCNA-D150E that spontaneously fall off DNA. In contrast, the elg1Δ mutator phenotype is exacerbated by PCNA mutants that accumulate on DNA due to enhanced electrostatic PCNA-DNA interactions. Epistasis analysis suggests that PCNA over-accumulation on DNA interferes with both MMR and MMR-independent process(es). In elg1Δ, over-retained PCNA hyper-recruits the Msh2-Msh6 mismatch recognition complex through its PCNA-interacting peptide motif, causing accumulation of MMR intermediates. Our results suggest that PCNA retention controlled by the Elg1 complex is critical for efficient MMR: PCNA needs to be on DNA long enough to enable MMR, but if it is retained too long it interferes with downstream repair steps.

Prevalence of elevated microsatellite alterations at selected tetranucleotide repeats in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) prognosis remains poor even after complete resection owing to no valuable biomarkers for recurrence and chemosensitivity. Tumors not expressing MSH3 show elevated microsatellite alterations at selected tetranucleotide repeats (EMAST). EMAST reportedly occurs in several tumors. In colorectal cancer (CRC), EMAST was reportedly correlated with 5-fluorouracil (5-FU) sensitivity. However, EMAST prevalence in PDAC and its significance as a prognostic biomarker are unknown. This study aimed to investigate EMAST prevalence in PDAC and the associations between EMAST and pathological factors, EMAST and prognosis, and EMAST and MSH3 expression via immunohistochemistry (IHC). We assessed 40 PDAC patients undergoing surgery. Genomic DNA was extracted from tumors and normal tissues. EMAST and microsatellite instability-high (MSI-H) were analyzed using five polymorphic tetranucleotide markers and five mononucleotide markers, respectively. Tumor sections were stained for MSH3, and staining intensity was evaluated via the Histoscore (H-score). Eighteen of 40 (45%) PDAC patients were EMAST-positive; however, none were MSI-H-positive. Clinicopathological characteristics including overall survival (OS) and recurrence-free survival (RFS) were not significantly different between EMAST-positive and EMAST-negative patients (P = 0.45, 0.98 respectively). IHC was performed to evaluate MSH3 protein expression levels for the PDAC tissue specimens. H-scores of EMAST-positive patients ranged from 0 to 300 (median, 40) and those of EMAST-negative patients ranged from 0 to 300 (median, 170). MSH3 protein was not significantly downregulated in EMAST-positive patients (P = 0.07). This study is a preliminary study and the number of cases investigated was small, and thus, study of a larger cohort will reveal the clinical implication of EMAST.