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Cancers of the gastrointestinal tract are a major cause of morbidity and mortality globally and, not surprisingly, there has been intense interest in identifying prognostic and predictive markers for new targeted therapies. The cancers of the gastrointestinal tract are unique in so completely exemplifying many of the key features of biomarker profiling across all solid cancers. Firstly, they involve the assessment of many individual biomarkers of many types, with both genomic and proteomic markers requiring assessment for complete profiling. Secondly, the interpretation of one biomarker is often critically dependent on the presence or absence of other biomarkers. And finally, this vast array of testing must be delivered often on very small samples. Here, we review the clinically-validated molecular targets in carcinomas of the esophagus, stomach, colorectum and anal canal, and in gastrointestinal stromal tumors, and discuss a handful of markers which are likely to be of use in the future. [ABSTRACT FROM AUTHOR]

MLH1 plays a critical role in DNA mismatch repair and genome maintenance. MLH1 deficiency promotes cancer development and progression, but the mechanism underlying MLH1 regulation remains enigmatic. In this study, we demonstrated that MLH1 protein is degraded by the ubiquitin-proteasome system and have identified vital cis-elements and trans-factors involved in MLH1 turnover. We found that the region encompassing the amino acids 516 to 650 is crucial for MLH1 degradation. The mismatch repair protein PMS2 may shield MLH1 from degradation as it binds to the MLH1 segment key to its turnover. Furthermore, we have identified the E3 ubiquitin ligase UBR4 and the deubiquitylase USP5, which oppositely modulate MLH1 stability. In consistence, UBR4 or USP5 deficiency affects the cellular response to nucleotide analog 6-TG, supporting their roles in regulating mismatch repair. Our study has revealed important insights into the regulatory mechanisms underlying MLH1 proteolysis, critical to DNA mismatch repair related diseases. [ABSTRACT FROM AUTHOR]

• Transcriptome profiling of Indian cassava is done for the first time here under drought stress conditions. • Field screening of clones confirmed the reduction in growth and yield under drought stress conditions. • Transcriptome profiling of tubers/starchy storage roots of cassava revealed novel genes involved in drought tolerance of cassava. • Among the differentially expressed genes under drought stress conditions in the tubers, aquaporins, growth regulator related genes and calcium binding and mineral transfer-related genes are prominent. • Antioxidant enzymes like glutathione peroxidase and starch/sucrose metabolic pathway related genes were upregulated in the tolerant clone 8S501 besides some common genes. Cassava (Manihot esculenta Carntz) is the major starchy root crop grown in the tropical parts of the world. Two cassava clones contrasting for their drought tolerance capacity (8S501- drought tolerant and H97-drought sensitive) were selected and grown in the field for studying the agronomical and molecular changes in both the clones under drought stress conditions. A reduction in the number of tuberous roots per plant, tuberous root length, average single tuber weight and tuber yield per plant was observed under drought stress when compared with the control conditions and also significantly lower mean values were observed for the sensitive clone H97 than that of the tolerant clone 8S501 when measured at 60 days of stress. Transcriptome sequencing of the tuber tissues of the two clones was done at 60 days of imposing drought stress. Different genes belonging to various groups like aquaporins, transporter genes, growth regulator related genes, calcium binding and mineral transfer-related genes, transferase genes and transcription factors were differentially expressed in the tubers under drought stress. Comparative transcriptomics revealed that acyl-CoA oxidase, primary-amine oxidase, glutathione peroxidase, vesicle transport, late embryogenesis abundant protein related, histone demethylase, BR-signaling kinase, palmitoyltransferase, DNA mismatch repair protein, heat shock protein related, starch/sucrose metabolic pathway related and MFS transporter genes were differentially expressed in the tubers of the tolerant clone 8S501 relative to the susceptible clone H97. [ABSTRACT FROM AUTHOR]

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 N3 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. [ABSTRACT FROM AUTHOR]

The approved standard dose of pembrolizumab (200 mg administrated every 3 weeks) for cancer treatment imposes a significant financial burden on patients. However, no study has analyzed the clinical outcomes of low-dose pembrolizumab among individuals diagnosed with gynecologic cancer. The primary objective of this study was to assess the effectiveness and safety of a low-dose pembrolizumab regimen in real-world clinical practice. We retrospectively assessed the efficacy and safety data of patients with gynecologic malignancies who received pembrolizumab between 2017 and 2022 at Kaohsiung Chang Gung Memorial Hospital. Furthermore, we conducted a comparative analysis of the objective response rate (ORR) and progression-free survival (PFS) between patients with deficient mismatch repair (dMMR) and proficient MMR (pMMR). A total of thirty-nine patients were included and received pembrolizumab at fixed dosages of 50 mg (5.1%), 100 mg (84.6%) and 200 mg (10.3%) per cycle. Compared to the pMMR group, the dMMR group exhibited a tendency toward improved ORR (45.5% vs. 13.0%, p = 0.074), and notably, the median duration of response remained unreached. There was no significant difference in PFS between the dMMR and pMMR groups; however, the patients with dMMR in tumor tissue had a trend of better survival (p = 0.079). Incidence of immune-related adverse events (irAEs) of any grade was observed in 13 patients (33.3%), with 3 individuals (7.7%) experiencing grade 3 or 4 events. Low-dose pembrolizumab may be a cost-effective and safe treatment option without compromising clinical outcomes in patients with refractory gynecologic cancers. [ABSTRACT FROM AUTHOR]

Unresectable locally advanced head and neck cancer (LAHNSCC) presents high heterogeneity1, which has yet to be elucidated at the molecular level. Consequently, it hampers the guidance of standard chemoradiotherapy (ChRT) and the implementation of precision medicine. Our study aims at assessing intratumor heterogeneity (ITH) and tumor evolution by examining the mutation profiles from WES in paired tissue and plasma both at baseline and relapse. Primary tumor (PT) biopsies from 61 unresectable LAHNSCC patients at baseline, along with parallel biopsies at relapse and paired plasma samples (baseline and relapse) from the INCLIVA Biobank's Head and Neck Cancer Collection were obtained. Patients provided informed consent, and the study received ethical approval from our Institutional review board. WES of the collected samples was performed on a HiSeq 3000 (Illumina) using the KAPA HyperExome (Roche) panel. UMI barcodes were added to correct background noise. Samples were selected based on a minimum average coverage (50x for WBC, 100x for FFPE and 250x for plasma samples). Only patients with White Blood Cells (WBCs) and plasma samples were included. Single-Nucleotide Variants (SNV), Copy Number Variants (CNVs) and small insertions and deletions (Indel) were called with an in-house bioinformatics pipeline. Minimum Variant allele frequencies (VAF) were set to 0.1% and 10% for plasma and tissue samples, respectively. Oncogenic somatic variants and related treatments were identified and annotated with OncoKB and Ensembl v107. Functional enrichment was performed using the cluster Profiler 4.6.2 R package and databases org.Hs.eg.db 3.16.0 and MSigDB 2023.1. A total of 37 unresectable LAHNSCC cases meeting quality criteria were included in our analysis (Figure 1). Clinicopathologic characteristics and treatment details are given in Table 1. [Display omitted] In Figure 2, we've employed an oncoprint to depict the WES results, showing the genomic status of the 58 most frequently mutated genes (CNVs and In/Del) in paired samples. [Display omitted] To discern the baseline genomic heterogeneity, we compared the WES results between a single biopsy of the PT and the paired baseline ctDNA from 30 patients. Among all the SNVs and In/Del mutations, an average of 88.2% of events displayed discrepancies between PT and ctDNA. Significantly, up to 67% of oncogenic mutations were exclusively identified in ctDNA. To assess ITH at relapse, we analyzed WES results from paired relapsed tissue biopsies and ctDNA (n=13). Our observations revealed a median concordance of 12.50%, with up to 58% of oncogenic mutations exclusively detected in plasma. To elucidate tumor evolution, we compared WES results of PT vs. relapsed tissue biopsies (n=10) and WES results of baseline ctDNA vs. ctDNA at relapse (n=37). When focusing on tissue analysis, a median concordance of 40% was observed, with up to 38% of oncogenic mutations exclusively identified in the relapsed tissue biopsy. In contrast, focusing on plasma results, discordance was higher, with only a 10.34% median concordance and up to 50% of oncogenic mutations found only in plasma at relapse. In our WES analysis of WBC, we identified pathogenic germline variants in 43.2% (16/37) of cases, highlighting mutations in BRCA2, CHEK2 and KIT as potentially actionable. No enrichment of APOBEC signatures or apoptosis pathways were detected. However, further analysis of defective DNA mismatch repair signatures and treatment-related signatures is currently underway. Our findings confirm the significant ITH in LAHNSCC, characterized by limited concordance between tissue and plasma mutational profiles. WES of ctDNA proves more effective at capturing this ITH, uncovering mutations absent in tissue profiles. Additionally, ctDNA-based WES enhances mutation detection during LAHNSCC relapse, offering valuable insights into resistance mechanisms to ChRT and enabling the potential use of targeted therapies to address these challenges. [ABSTRACT FROM AUTHOR]

Germline and somatic mutations cause various diseases, including cancer. Clinical applications of genome editing are keenly anticipated, since it can cure genetic diseases. Recently, we reported that a 5′-tailed duplex (TD), consisting of an approximately 80-base editor strand oligodeoxyribonucleotide and a 35-base assistant strand oligodeoxyribonucleotide, could edit a target gene on plasmid DNA and correct a single-base substitution mutation without an artificial nuclease in human cells. In this study, we assessed the ability of the TD to correct base substitution mutations located consecutively or separately, and deletion and insertion mutations. A TD with an 80-base editor strand was co-introduced into human U2OS cells with plasmid DNA bearing either a wild-type or mutated copepod green fluorescent protein (copGFP) gene. Among the mutations, three-base consecutive substitutions were efficiently repaired. The correction efficiencies of deletion mutations were similar to those of substitution mutations, and two to three times higher than those of insertion mutations. Up to three-base substitution, deletion, and insertion mutations were excellent targets for correction by TDs. These results suggested that the TDs are useful for editing disease-causing genes with small mutations. [ABSTRACT FROM AUTHOR]

Bacterial extracellular DNA (eDNA) is important for the development of virtually all biofilms, their architecture, and their resistance to host defense. DNase I and DNase1L3 are important host immune strategies to prevent biofilm formation. eDNA in biofilms can adopt a Z-form, an alternative configuration of DNA that increases biofilm rigidity and is maintained by DNA-binding proteins. Z-DNA is resistant to DNase I, suggesting that bacteria have learned to circumvent certain host innate immune strategies by changing their eDNA physical form. DNA sensors cGAS/STING are involved in immune responses against Porphyromonas gingivalis biofilms in periodontitis and in the vicious cycle that maintains dysbiotic microbiota and chronic intestinal inflammation in IBD suggesting a role in recognizing eDNA in biofilms. eDNA-binding proteins are essential for biofilm integrity and eDNA/protein complexes are pathogen-associated molecular patterns (PAMPs) recognized by pattern recognition receptors (PRRs) like Toll-like receptors (TLRs). Many reports support the working hypothesis that eDNA is a universal biofilm-associated PAMP and that targeting eDNA might represent a novel therapeutic strategy against recurrent/chronic bacterial infections that generate biofilms. Identifying the role of extracellular DNA in mammalian immune recognition of biofilms can improve our understanding of innate immune responses to bacterial infections; it may also help in the discovery of novel therapeutic targets against certain chronic/recurrent/antibiotic-resistant infections and autoimmunity. Historically, the study of innate immune detection of bacterial infections has focused on the recognition of pathogen-associated molecular patterns (PAMPs) from bacteria growing as single cells in planktonic phase. However, over the past two decades, studies have highlighted an adaptive advantage of bacteria: the formation of biofilms. These structures are complex fortresses that stand against a hostile environment, including antibiotics and immune responses. Extracellular DNA (eDNA) is a crucial component of the matrix of most known biofilms. In this opinion article, I propose that eDNA is a universal PAMP that the immune system uses to recognize biofilms. Outstanding questions concern the discrimination between biofilm-associated eDNA and DNA from planktonic bacteria, the innate receptors involved, and the immune response to biofilms. [ABSTRACT FROM AUTHOR]

Gastrointestinal cancers are one of the most frequent cancers and a leading cause of cancer deaths worldwide. We provide an overview of the most important practice-changing trials that were either published or presented at the international scientific meetings in 2021–2023. Highlights included reports on three phase III trials (CONCORDE/PRODIGE 26, ARTDECO, and a study by Xu et al.) that evaluated dose escalation in the definitive setting for locally advanced oesophageal cancers, as well as two phase III trials that evaluated the role of chemotherapy (neo-AEGIS) and targeted therapy (NRG/RTOG 1010) in the neoadjuvant setting for adenocarcinoma oesophageal cancers or gastroesophageal junction cancer. CheckMate 577 evaluated nivolumab in patients who had residual pathological disease after neoadjuvant chemoradiation followed by complete resection. The use of radiation therapy for borderline and locally advanced pancreatic cancer is also discussed (SMART and CONKO-007 trials). Stereotactic body radiation therapy followed by sorafenib was compared to sorafenib alone in patients with hepatocellular carcinoma in the NRG/RTOG 1112 study. New options in the management of rectal cancer are emerging such as total neoadjuvant treatment (PRODIGE 23, RAPIDO, PROSPECT), organ preservation (OPRA, OPERA), and the role of immunotherapy in patients with DNA mismatch-repair deficient/microsatellite instability. Finally, preliminary results of the ACT 4 trial that evaluated de-escalation in anal cancer are presented. [ABSTRACT FROM AUTHOR]

The effect of genomic factors on the response of patients with esophageal squamous cell carcinoma (ESCC) to neoadjuvant chemoradiotherapy (nCRT), as well as how nCRT influences the genome and transcriptome of ESCC, remain largely unknown. In total, 137 samples from 57 patients with ESCC undergoing nCRT were collected and subjected to whole-exome sequencing and RNA sequencing analysis. Genetic and clinicopathologic factors were compared between the patients achieving pathologic complete response and patients not achieving pathologic complete response. Genomic and transcriptomic profiles before and after nCRT were analyzed. Codeficiency of the DNA damage repair and HIPPO pathways synergistically sensitized ESCC to nCRT. nCRT induced small INDELs and focal chromosomal loss concurrently. Acquired INDEL% exhibited a decreasing trend with the increase of tumor regression grade (P =.06, Jonckheere's test). Multivariable Cox analysis indicated that higher acquired INDEL% was associated with better survival (adjusted hazard ratio [aHR], 0.93; 95% CI, 0.86-1.01; P =.067 for recurrence-free survival [RFS]; aHR, 0.86; 95% CI, 0.76-0.98; P =.028 for overall survival [OS], with 1% of acquired INDEL% as unit). The prognostic value of acquired INDEL% was confirmed by the Glioma Longitudinal AnalySiS data set (aHR, 0.95; 95% CI, 0.902-0.997; P =.037 for RFS; aHR, 0.96; 95% CI, 0.917-1.004; P =.076 for OS). Additionally, clonal expansion degree was negatively associated with patient survival (aHR, 5.87; 95% CI, 1.10-31.39; P =.038 for RFS; aHR, 9.09; 95% CI, 1.10-75.36; P =.041 for OS, with low clonal expression group as reference) and also negatively correlated with acquired INDEL% (Spearman ρ = –0.45; P =.02). The expression profile was changed after nCRT. The DNA replication gene set was downregulated, while the cell adhesion gene set was upregulated after nCRT. Acquired INDEL% was negatively correlated with the enrichment of the DNA replication gene set (Spearman ρ = –0.56; P =.003) but was positively correlated with the enrichment of the cell adhesion gene set (Spearman ρ = 0.40; P =.05) in posttreatment samples. nCRT remodels the genome and transcriptome of ESCC. Acquired INDEL% is a potential biomarker to indicate the effectiveness of nCRT and radiation sensitivity. [ABSTRACT FROM AUTHOR]

[Display omitted] Epstein-Barr virus (EBV) is the oncogenic driver of multiple cancers. However, the underlying mechanism of virus-cancer immunological interaction during disease pathogenesis remains largely elusive. Here we reported the first comprehensive proteogenomic characterization of natural killer/T-cell lymphoma (NKTCL), a representative disease model to study EBV-induced lymphomagenesis, incorporating genomic, transcriptomic, and in-depth proteomic data. Our multi-omics analysis of NKTCL revealed that EBV gene pattern correlated with immune-related oncogenic signaling. Single-cell transcriptome further delineated the tumor microenvironment as immune-inflamed, -deficient, and -desert phenotypes, in association with different setpoints of cancer-immunity cycle. EBV interacted with transcriptional factors to provoke GPCR interactome (GPCRome) reprogramming. Enhanced expression of chemokine receptor-1 (CCR1) on malignant and immunosuppressive cells modulated virus-cancer interaction on microenvironment. Therapeutic targeting CCR1 showed promising efficacy with EBV eradication, T-cell activation, and lymphoma cell killing in NKTCL organoid. Collectively, our study identified a previously unknown GPCR-mediated malignant progression and translated sensors of viral molecules into EBV-specific anti-cancer therapeutics. [ABSTRACT FROM AUTHOR]

Peritoneal metastasis (PM) is a fatal state of colorectal cancer, and only a few patients may benefit from systemic chemotherapy. Although hyperthermic intraperitoneal chemotherapy (HIPEC) brings hope for affected patients, the drug development and preclinical evaluation of HIPEC are seriously lagging behind, mainly due to the lack of an ideal in vitro PM model that makes drug development over-reliant on expensive and inefficient animal experiments. This study developed an in vitro colorectal cancer PM model [microvascularized tumor assembloids (vTA)] based on an assembly strategy of endothelialized microvessels and tumor spheroids. Our data showed that the in vitro perfusion cultured vTA could maintain a similar gene expression pattern to their parental xenografts. Also, the drug penetration pattern of the in vitro HIPEC in vTA could mimic the drug delivery behavior in tumor nodules during in vivo HIPEC. More importantly, we further confirmed the feasibility of constructing a tumor burden-controlled PM animal model using vTA. In conclusion, we propose a simple and effective strategy to construct physiologically simulated PM models in vitro , thus providing a basis for PM-related drug development and preclinical evaluation of locoregional therapies. This study developed an in vitro colorectal cancer peritoneal metastasis (PM) model based on microvascularized tumor assembloids (vTA) for drug evaluation. With perfusion culture, vTA could maintain a similar gene expression pattern and tumor heterogeneity to their parental xenografts. And the drug penetration pattern in vTA was similar to the drug delivery behavior in tumor nodules under in vivo treatment. Moreover, vTA was more conducive to construct PM animal models with controllable tumor burden. In conclusion, the construction of vTA could provide a new strategy for the PM-related drug development and preclinical evaluation of locoregional therapies. [Display omitted] [ABSTRACT FROM AUTHOR]

• The MMR booster vaccine demonstrated long-term immunogenicity in the majority of children with JIA. • Percentages of protective antibody levels against measles were lower in bDMARD users compared to non-bDMARD users. • Clinicians should consider to measure antibody levels at least five years after MMR booster in bDMARD users and advice an extra booster accordingly. Vaccines, especially live attenuated vaccines, in children with JIA pose a great challenge due to both potential lower immunogenicity and safety as a result of immunosuppressive treatment. For many years, in the Netherlands, JIA patients receive a measles-mumps-rubella (MMR) booster vaccine at the age of nine years as part of the national immunization program. To study long-term humoral immunoprotection in a large cohort of JIA patients who received the MMR booster vaccine while being treated with immunomodulatory therapies at the Wilhelmina Children's Hospital in Utrecht, the Netherlands. MMR-specific IgG antibody concentrations in stored serum samples of vaccinated JIA patients were determined with chemiluminescent microparticle immunoassays (CMIA). Samples were analyzed five years after MMR booster vaccination and at last available follow-up visit using both crude and adjusted analyses. Additional clinical data were collected from electronic medical records. In total, 236 samples from 182 patients were analyzed, including 67 samples that were available five years post-vaccination, and an additional 169 samples available from last visits with a median duration after vaccination of 6.9 years (IQR: 2.8–8.8). Twenty-eight patients were using biologic disease-modifying antirheumatic drugs (bDMARDS) of whom 96% anti-TNF agents and 4% tocilizumab. Percentages of protective antibody levels against measles after five years were significantly lower for patients who used bDMARD therapy at vaccination compared to patients who did not: 60% versus 86% (P = 0.03). For mumps (80% versus 94%) and rubella (60% versus 83%) this difference did not reach statistical significance (P = 0.11 and P = 0.07, respectively). Antibody levels post-vaccination decreased over time, albeit not significantly different between bDMARD users and non-bDMARD users. The MMR booster vaccine demonstrated long-term immunogenicity in the majority of children with JIA from a large cohort, although lower percentages of protective measles antibody levels were observed in bDMARD users. Hence, it might be indicated to measure antibody levels at least five years after MMR booster vaccination in the latter group and advice an extra booster accordingly. [ABSTRACT FROM AUTHOR]

Mismatch repair-deficient (dMMR) tumors displaying microsatellite instability (MSI) represent a paradigm for the success of immune checkpoint inhibitor (ICI)-based immunotherapy, particularly in patients with metastatic colorectal cancer (mCRC). However, a proportion of patients with dMMR/MSI mCRC exhibit resistance to ICI. Identification of tools predicting MSI mCRC patient response to ICI is required for the design of future strategies further improving this therapy. We combined high-throughput DNA and RNA sequencing of tumors from 116 patients with MSI mCRC treated with anti-programmed cell death protein 1 ± anti-cytotoxic T-lymphocyte-associated protein 4 of the NIPICOL phase II trial (C1, NCT03350126, discovery set) and the ImmunoMSI prospective cohort (C2, validation set). The DNA/RNA predictors whose status was significantly associated with ICI status of response in C1 were subsequently validated in C2. Primary endpoint was progression-free survival by immune RECIST (iRECIST) (iPFS). Analyses showed no impact of previously suggested DNA/RNA indicators of resistance to ICI, e.g. MSIsensor score, tumor mutational burden, or specific cellular and molecular tumoral contingents. By contrast, iPFS under ICI was shown in C1 and C2 to depend both on a multiplex MSI signature involving the mutations of 19 microsatellites hazard ratio cohort C2 (HR C2) = 3.63; 95% confidence interval (CI) 1.65-7.99; P = 1.4 × 10–3] and the expression of a set of 182 RNA markers with a non-epithelial transforming growth factor beta (TGFB)-related desmoplastic orientation (HR C2 = 1.75; 95% CI 1.03-2.98; P = 0.035). Both DNA and RNA signatures were independently predictive of iPFS. iPFS in patients with MSI mCRC can be predicted by simply analyzing the mutational status of DNA microsatellite-containing genes in epithelial tumor cells together with non-epithelial TGFB-related desmoplastic RNA markers. • ChallengES tumor mutation burden (TMB) and MSIsensor for predicting response to immunotherapy in MSI mCRC. • Demonstration that MSI misdiagnoses lead to a false ability of TMB and MSIsensor to predict ICI resistance in MSI mCRC. • Identification and validation of DNA and RNA signatures predictive of response to ICI in these patients with mCRC MSI. • Evidence for a fibrosis signature associated with functional ICI resistance in MSI mCRC. [ABSTRACT FROM AUTHOR]

Transcription-coupled repair (TCR) is a subpathway of nucleotide excision repair (NER) that is regulated by multiple facilitators, such as Rad26, and repressors, such as Rpb4 and Spt4/Spt5. How these factors interplay with each other and with core RNA polymerase II (RNAPII) remains largely unknown. In this study, we identified Rpb7, an essential RNAPII subunit, as another TCR repressor and characterized its repression of TCR in the AGP2, RPB2, and YEF3 genes, which are transcribed at low, moderate, and high rates, respectively. The Rpb7 region that interacts with the KOW3 domain of Spt5 represses TCR largely through the same common mechanism as Spt4/Spt5, as mutations in this region mildly enhance the derepression of TCR by spt4Δ only in the YEF3 gene but not in the AGP2 or RPB2 gene. The Rpb7 regions that interact with Rpb4 and/or the core RNAPII repress TCR largely independently of Spt4/Spt5, as mutations in these regions synergistically enhance the derepression of TCR by spt4Δ in all the genes analyzed. The Rpb7 regions that interact with Rpb4 and/or the core RNAPII may also play positive roles in other (non-NER) DNA damage repair and/or tolerance mechanisms, as mutations in these regions can cause UV sensitivity that cannot be attributed to derepression of TCR. Our study reveals a novel function of Rpb7 in TCR regulation and suggests that this RNAPII subunit may have broader roles in DNA damage response beyond its known function in transcription. [ABSTRACT FROM AUTHOR]

De novo somatic mutations are difficult to analyze because of their random nature. Apart from the few clonally amplified mutations, the far majority differs from cell to cell. Recent advances in single-cell, single-molecule and high-depth sequencing have shown that somatic mutations accumulate with age in virtually all tissues. Mutations arise through errors during replication or repair of damaged DNA. Age-related somatic mutation accumulation is inevitable due to the lack of an evolutionary advantage in protecting genome sequence integrity far beyond the period of first reproduction. Somatic mutation rate might be mitigated by adopting genome maintenance strategies of (i) extremely long-lived animals; (ii) species living under high genotoxic conditions; or (iii) stem or germ cells. Genomes are inherently unstable and require constant DNA repair to maintain their genetic information. However, selective pressure has optimized repair mechanisms in somatic cells only to allow transmitting genetic information to the next generation, not to maximize sequence integrity long beyond the reproductive age. Recent studies have confirmed that somatic mutations, due to errors during genome repair and replication, accumulate in tissues and organs of humans and model organisms. Here, we describe recent advances in the quantitative analysis of somatic mutations in vivo. We also review evidence for or against a possible causal role of somatic mutations in aging. Finally, we discuss options to prevent, delay or eliminate de novo , random somatic mutations as a cause of aging. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Adoptive cell therapy (ACT) using specific immune cells and stem cells has emerged as a promising treatment option that could complement traditional cancer therapies in the future. In particular, tumor-infiltrating lymphocytes (TILs) have been shown to be effective against solid tumors in various clinical trials. Despite the enormous disease burden and large number of premature deaths caused by colorectal cancer (CRC), studies on TILs isolated from tumor tissue of patients with CRC are still rare. To date, studies on ACT often lack controlled and comparable expansion processes as well as selected ACT-relevant T-cell populations. We describe a procedure for generating patient-specific TILs, which are prerequisites for clinical trials of ACT in CRC. The manufacturing and characteristics of these TILs differ in important modalities from TILs commonly used for this therapeutic approach. Tumor tissue samples were obtained from 12 patients undergoing surgery for primary CRC, predominantly with low microsatellite instability (pMMR-MSI-L). Tumors in the resected specimens were examined pathologically, and an approved volume of tumor tissue was transferred to a disposable perfusion bioreactor. Tissue samples were subjected to an automatically controlled and highly reproducible cultivation process in a GMP-conform, closed perfusion bioreactor system using starting medium containing interleukin-2 and interleukin-12. Outgrowth of TIL from tissue samples was initiated by short-term supplementation with a specific activation cocktail. During subsequent expansion, TILs were grown in interleukin-2–enriched medium. Expansion of TILs in a low-scaled, two-phase process in the Zellwerk ZRP bioreactor under hyperoxic conditions resulted in a number of approximately 2 × 109 cells. The expanded TILs consisted mainly (73%) of the ACT-relevant CD3+/CD8+ effector memory phenotype (CD45RO+/CCR7−). TILs harvested under these conditions exhibited high functional potential, which was confirmed upon nonspecific stimulation (interferon-γ, tumor necrosis factor-α cytokine assay) [ABSTRACT FROM AUTHOR]

The DNA mismatch repair (MMR) system is a major DNA repair system that suppresses both inherited and sporadic cancers in humans. In eukaryotes, the MutSa-dependent and MutSβ-dependent MMR pathways correct DNA polymerase errors. Here, we investigated these two pathways on a whole genome level in Saccharomyces cerevisiae. We found that inactivation of MutSa-dependent MMR increases the genomewide mutation rate by ~17-fold and loss of MutSβ-dependent MMR elevates the genome-wide mutation rate by ~4-fold. We also found that MutSa-dependent MMR does not show a preference for protecting coding or noncoding DNA from mutations, whereas MutSβ-dependent MMR preferentially protects noncoding DNA from mutations. The most frequent mutations in the msh6Δ strain are C>T transitions, whereas 1- to 6-bp deletions are the most common genetic alterations in the msh3Δ strain. Strikingly, MutSa-dependent MMR is more important than MutSβ-dependent MMR for protection from 1-bp insertions, while MutSβ-dependent MMR has a more critical role in the defense against 1-bp deletions and 2- to 6-bp indels. We also determined that a mutational signature of yeast MSH6 loss is similar to mutational signatures of human MMR deficiency. Furthermore, our analysis showed that compared to other 50-NCN-30 trinucleotides, 50-GCA-30 trinucleotides are at the highest risk of accumulating C>T transitions at the central position in the msh6Δ cells and that the presence of a G/A base at the -1 position is important for the efficient MutSa-dependent suppression of C>T transitions. Our results highlight key differences between the roles of the MutSa-dependent and MutSβ-dependent MMR pathways. [ABSTRACT FROM AUTHOR]

Lynch syndrome (LS) is one of the most prevalent hereditary cancer syndromes in humans and accounts for some 3% of unselected patients with colorectal or endometrial cancer and 10%–15% of those with DNA mismatch repair–deficient tumors. Previous studies have established the genetic basis of LS predisposition, but there have been significant advances recently in the understanding of the molecular pathogenesis of LS tumors, which has important implications in clinical management. At the same time, immunotherapy has revolutionized the treatment of advanced cancers with DNA mismatch repair defects. We aim to review the recent progress in the LS field and discuss how the accumulating epidemiologic, clinical, and molecular information has contributed to a more accurate and complete picture of LS, resulting in genotype- and immunologic subtype–specific strategies for surveillance, cancer prevention, and treatment. [ABSTRACT FROM AUTHOR]

DNA mismatch repair (MMR) in eukaryotes is believed to occur post-replicatively, wherein nicks or gaps in the nascent DNA strand are suggested to serve as strand discrimination signals. However, how such signals are generated in the nascent leading strand has remained unclear. Here we examine the alternative possibility that MMR occurs in conjunction with the replication fork. To this end, we utilize mutations in the PCNA interacting peptide (PIP) domain of the Pol3 or Pol32 subunit of DNA polymerase d (Pold) and show that these pip mutations suppress the greatly elevated mutagenesis in yeast strains harboring the pol3-01 mutation defective in Pold proofreading activity. And strikingly, they suppress the synthetic lethality of pol3-01 pol2-4 double mutant strains, which arises from the vastly enhanced mutability due to defects in the proofreading functions of both Pold and Pole. Our finding that suppression of elevated mutagenesis in pol3-01 by the Pold pip mutations requires intact MMR supports the conclusion that MMR operates at the replication fork in direct competition with other mismatch removal processes and with extension of synthesis from the mispair by Pold. Furthermore, the evidence that Pold pip mutations eliminate almost all the mutability of pol2-4 msh2Δ or pol3-01 pol2-4 adds strong support for a major role of Pold in replication of both the leading and lagging DNA strands. [ABSTRACT FROM AUTHOR]

• HRD test is widely used for administrating PARPi chemotherapy in breast cancer. • Existing methods are either experimentally complicated or commercially expensive. • Low-pass WGS based HRD detection method performs well in breast cancer. • Present an HRD test with high accuracy, ease of operation, and acceptable cost. Homologous recombination repair deficiency (HRD) results in a defect in DNA repair and is a frequent driver of tumorigenesis. Poly(ADP-ribose) polymerase inhibitors (PARPi) or platinum-based therapies have increased theraputic effectiveness when treating HRD positive cancers. For breast cancer and ovairan cancer HRD companion diagnostic tests are commonly used. However, the currently used HRD tests are based on high-depth genome sequencing or hybridization-based capture sequencing, which are technically complex and costly. In this study, we modified an existing method named shallowHRD, which uses low-pass whole genome sequencing (WGS) for HRD detection, and estimated the performance of the modified shallowHRD pipeline. Our shallowHRD pipeline achieved an AUC of 0.997 in simulated low-pass WGS data, with a sensitivity of 0.981 and a specificity of 0.964; and achieved a higher HRD risk score in clinical BRCA-deficient breast cancer samples (p = 5.5 × 10−5, compared with BRCA-intact breast cancer samples). We also estimated the limit of detection the shallowHRD pipeline could accurately predict HRD status with a minimum sequencing depth of 0.1 ×, a tumor purity of > 20%, and an input DNA amount of 1 ng. Our study demostrates using low-pass sequencing, HRD status can be determined with high accuracy using a simple approach with greatly reduced cost. [ABSTRACT FROM AUTHOR]

• Many genetic associations with vaccine immunogenicity have been identified. • Most genetic associations identified with Hepatitis B, MMR, and influenza vaccines. • Genetic associations were identified with 6 adverse events following immunization. • Vaccinomics is a growing field with great potential but need for investment. • Progress in vaccinomics could improve vaccine safety and effectiveness. This scoping review summarizes a key aspect of vaccinomics by collating known associations between heterogeneity in human genetics and vaccine immunogenicity and safety. We searched PubMed for articles in English using terms covering vaccines routinely recommended to the general US population, their effects, and genetics/genomics. Included studies were controlled and demonstrated statistically significant associations with vaccine immunogenicity or safety. Studies of Pandemrix®, an influenza vaccine previously used in Europe, were also included, due to its widely publicized genetically mediated association with narcolepsy. Of the 2,300 articles manually screened, 214 were included for data extraction. Six included articles examined genetic influences on vaccine safety; the rest examined vaccine immunogenicity. Hepatitis B vaccine immunogenicity was reported in 92 articles and associated with 277 genetic determinants across 117 genes. Thirty-three articles identified 291 genetic determinants across 118 genes associated with measles vaccine immunogenicity, 22 articles identified 311 genetic determinants across 110 genes associated with rubella vaccine immunogenicity, and 25 articles identified 48 genetic determinants across 34 genes associated with influenza vaccine immunogenicity. Other vaccines had fewer than 10 studies each identifying genetic determinants of their immunogenicity. Genetic associations were reported with 4 adverse events following influenza vaccination (narcolepsy, GBS, GCA/PMR, high temperature) and 2 adverse events following measles vaccination (fever, febrile seizure). This scoping review identified numerous genetic associations with vaccine immunogenicity and several genetic associations with vaccine safety. Most associations were only reported in one study. This illustrates both the potential of and need for investment in vaccinomics. Current research in this field is focused on systems and genetic-based studies designed to identify risk signatures for serious vaccine reactions or diminished vaccine immunogenicity. Such research could bolster our ability to develop safer and more effective vaccines. [ABSTRACT FROM AUTHOR]

[Display omitted] • Cell lines with MMR and proofreading deficiency were constructed using the CRISPR technology for reference samples. • Paired tumor–normal reference samples close to clinical specimens were prepared. • A reliable process to determine high-confidence region and positive variants was designed. • A multi-laboratory study was conducted to evaluate reproducibility and accuracy of participating panels. • Potential sources of false-discovery were explored for assay optimization. Clinical precision oncology increasingly relies on accurate genome-wide profiling using large panel next generation sequencing; however, difficulties in accurate and consistent detection of somatic mutation from individual platforms and pipelines remain an open question. To obtain paired tumor–normal reference materials that can be effectively constructed and interchangeable with clinical samples, and evaluate the performance of 56 panels under routine testing conditions based on the reference samples. Genes involved in mismatch repair and DNA proofreading were knocked down using the CRISPR-Cas9 technology to accumulate somatic mutations in a defined GM12878 cell line. They were used as reference materials to comprehensively evaluate the reproducibility and accuracy of detection results of oncopanels and explore the potential influencing factors. In total, 14 paired tumor–normal reference DNA samples from engineered cell lines were prepared, and a reference dataset comprising 168 somatic mutations in a high-confidence region of 1.8 Mb were generated. For mutations with an allele frequency (AF) of more than 5% in reference samples, 56 panels collectively reported 1306 errors, including 729 false negatives (FNs), 179 false positives (FPs) and 398 reproducibility errors. The performance metric varied among panels with precision and recall ranging from 0.773 to 1 and 0.683 to 1, respectively. Incorrect and inadequate filtering accounted for a large proportion of false discovery (including FNs and FPs), while low-quality detection, cross-contamination and other sequencing errors during the wet bench process were other sources of FNs and FPs. In addition, low AF (<5%) considerably influenced the reproducibility and comparability among panels. This study provided an integrated practice for developing reference standard to assess oncopanels in detecting somatic mutations and quantitatively revealed the source of detection errors. It will promote optimization, validation, and quality control among laboratories with potential applicability in clinical use. [ABSTRACT FROM AUTHOR]

• The recommended age of MMR vaccination changed in 2011 creating a natural experiment. • We observed no non-specific effect on all-cause infections due to MMR vaccination. • MMR vaccination was associated with fewer urinary tract infections. According to earlier studies, live vaccines like measles-mumps-rubella (MMR) vaccine could reduce also other infections than only the infections they are targeted against. This non-specific effect has been seen especially in studies in low-income countries and results from high-income countries have not been unambiguous. In 2011 Finland changed the recommended schedule for the first MMR vaccination from 18 months to 12 months of age. This change created a natural experiment for evaluating the potential non-specific effects. This is a retrospective nationwide register-based cohort study of Finnish children born between 2008 and 2012. Children were divided into two cohorts by age at MMR vaccination: children administered early MMR vaccination (11 through 12 months of age) and late MMR vaccination (18 through 19 months of age). Morbidity was evaluated during the main follow-up period (from 13 to 17 months of age) and before any MMR vaccination (3 to 10 months) and after all were vaccinated with MMR (20 to 35 months) as control follow-up periods. We analyzed all infections and did additional analyzes for urinary tract infections (UTI) and bronchitis. Injuries were analyzed as a control outcome. Early MMR vaccinated children (N = 79 949) had fewer infections compared to late MMR vaccinated (N = 60 965) during the main follow-up period. The incidence rate ratio (IRR) was 0.84 (95 % confidence interval (95 % CI) 0.81–0.87). However, similar differences were also observed during the control follow-up periods. MMR vaccinated children had less UTI in the main follow-up period (IRR 0.73, 0.60–0.89) but not in the control follow-up periods. When stratified by sex, the difference was observed among girls but not in boys. No clear evidence was found for non-specific effects in infectious diseases morbidity. However, there could be a nonspecific effect on UTI. Confirmation is needed from other studies, especially from high-income countries. [ABSTRACT FROM AUTHOR]

We show that the rates of single base substitutions, additions, and deletions across the nuclear genome are strongly increased in a strain harboring a mutator variant of DNA polymerase α combined with a mutation that inactivates the 3´-5´ exonuclease activity of DNA polymerase δ. Moreover, tetrad dissections attempting to produce a haploid triple mutant lacking Msh6, which is essential for DNA mismatch repair (MMR) of base•base mismatches made during replication, result in tiny colonies that grow very slowly and appear to be aneuploid and/or defective in oxidative metabolism. These observations are consistent with the hypothesis that during initiation of nuclear DNA replication, single-base mismatches made by naturally exonuclease-deficient DNA polymerase α are extrinsically proofread by DNA polymerase δ, such that in the absence of this proofreading, the mutation rate is strongly elevated. Several implications of these data are discussed, including that the mutational signature of defective extrinsic proofreading in yeast could appear in human tumors. • Errors by Pol α variant, Pol1-L868M, are proofread by Pol δ across the yeast genome. • Indels by Pol α in long homopolymer runs are preferentially repaired by Pol δ. • Haploid pol1-L868M pol3–5DV msh6Δ yeast approach error catastrophe. [ABSTRACT FROM AUTHOR]

As a consequence of global climate change, the increasing frequency of environmental disturbances and surplus oxidative stress experienced by coral reefs will likely contribute to phase shifts from stony to soft corals. Mitochondrial response to reactive oxygen species (ROS) -induced oxidative damage appears pivotal for bioenergetic adaptation and recovery during environmental stress, partly governed by mitochondrial DNA copy number. Unlike other animals, octocorals possess unique mitogenomes with an intrinsic DNA mismatch repair gene, the mtMutS, that is likely to have a role in mitochondrial response and mtDNA damage recovery. Yet, there is a general lack of stress response studies on octocorals from a mitochondrial perspective. Here we evaluate the mitochondrial response of the octocoral Sclerophytum sp. subjected to acute elevated temperature and low pH, and its putative competence to reverse oxidative mtDNA damage caused by exogenous agents like hydrogen peroxide (H 2 O 2). Temporal changes in mtDNA copy number and mtDNA damage and recovery were monitored. Both short-term thermal and low pH stress applied independently instigated mtDNA damage and affected mtDNA copy number differently, while mtMutS gene was significantly upregulated during low pH stress. mtDNA damage caused by H 2 O 2 insult was observed to be promptly reversed in Sclerophytum sp., and a higher mtDNA copy number was associated with lower mtDNA damage. These findings provide insights into the potential role of mtMutS gene in conferring resilience to octocorals, the relevance of mtDNA copy number, and emphasize the importance of better understanding the mitochondrial stress response of cnidarians in the context of climate change. • Octocorals possess unique mitochondrial genomes with an intrinsic DNA mismatch repair gene, mtMutS. • Short-term thermal and low pH stress cause mtDNA damage and variation in mtDNA copy number in the soft coral Sclerophytum sp. • mtMutS gene was significantly upregulated during low pH stress. • mtDNA damage caused by H 2 O 2 was quickly reversed, suggesting efficient mtMutS DNA repair activity. • Higher mtDNA copy number was associated with lower mtDNA damage. [ABSTRACT FROM AUTHOR]

Cancers frequently arise from a multi-stage successive acquisition of somatic (i.e. acquired) mutations. A limited number of these mutations may lead to a clonal expansion of the mutated cell population, without malignant transformation. Clonal hematopoiesis (CH) refers to somatic mutations leading to clonal expansion in the hematopoietic. Individuals with CH, including those with cancer, have a large increased risk of hematologic malignancy and worse overall survival. Additionally, CH is associated with an increased risk of numerous non-cancer adverse health outcomes including cardiovascular, cerebrovascular, pulmonary, endocrine, renal, and liver disease. There are limited prospective studies investigating the genesis and evolution of CH following cancer treatments using serial samples. We hypothesize that chemoradiation therapy is associated with increased CH mutations and these changes are associated with clinical outcomes. We undertook error-corrected duplex sequencing in DNA from blood collected prior to, and two timepoints following, chemoradiation for thoracic malignancies in 29 patients (n=87 samples). We applied a customized workflow to identify and examine the earliest changes in CH mutation count, clone size, and their association with clinical outcomes. Median participant age was 67 years, 76% (n=22) were male, and median follow-up was 3.9 years. The most mutated genes were DNMT3A, TET2, TP53, and ASXL1. We observed a two-fold increase in the number of mutations from pre-treatment to post-treatment in TP53, which differed from all other genes examined (p<0.001), and was driven by post-therapy selection for missense mutations. Mutational signature analysis demonstrated immunosuppression and defective DNA mismatch repair patterns. Among mutations detected pre- and post-treatment, we observed an increased clone size in 38% and a decreased clone size in 5% of TP53 mutations (odds ratio, 3.7; 95% CI, 1.75-7.84; p<0.001). Changes in mutation count and clone size were not observed in other genes. Individuals with an increase in the number of TP53 mutations following chemoradiation experienced worse overall survival (hazard ratio, 7.07; 95% CI, 1.50-33.46; p=0.014). Here we show an increase in the number of CH mutations and size of clones following chemoradiation therapy in patients with solid malignancy, specifically in DDR genes. Additionally, we found an association between increased TP53 mutations following therapy and worse survival. Future studies are needed to better understand which patients are most at risk for therapy associated CH and the mechanisms by which these acquired changes may impact clinical outcomes. [ABSTRACT FROM AUTHOR]

By replicating damaged nucleotides, error-prone DNA translesion synthesis (TLS) enables the completion of replication, albeit at the expense of fidelity. TLS of helix-distorting DNA lesions, that usually have reduced capacity of basepairing, comprises insertion opposite the lesion followed by extension, the latter in particular by polymerase ζ (Pol ζ). However, little is known about involvement of Pol ζ in TLS of non- or poorly-distorting, but miscoding, lesions such as O 6-methyldeoxyguanosine (O 6-medG). Using purified Pol ζ we describe that the enzyme can misincorporate thymidine opposite O 6-medG and efficiently extend from terminal mismatches, suggesting its involvement in the mutagenicity of O 6-medG. Surprisingly, O6-medG lesions induced by the methylating agent N -methyl- N' -nitro- N -nitrosoguanidine (MNNG) appeared more, rather than less, mutagenic in Pol ζ-deficient mouse embryonic fibroblasts (MEFs) than in wild type MEFs. This suggested that in vivo Pol ζ participates in non-mutagenic TLS of O 6-medG. However, we found that the Pol ζ-dependent misinsertions at O6-medG lesions are efficiently corrected by DNA mismatch repair (MMR), which masks the error-proneness of Pol ζ. We also found that the MNNG-induced mutational signature is determined by the adduct spectrum, and modulated by MMR. The signature mimicked single base substitution signature 11 in the catalogue of somatic mutations in cancer, associated with treatment with the methylating drug temozolomide. Our results unravel the individual roles of the major contributors to methylating drug-induced mutagenesis. Moreover, these results warrant caution as to the classification of TLS as mutagenic or error-free based on in vitro data or on the analysis of mutations induced in MMR-proficient cells. [Display omitted] • Polymerase ζ or a redundant polymerase extend DNA after Thymidine misincorporation opposite O6-methyldeoxyguanosine. • DNA mismatch repair is particularly efficient in resolving Polymerase ζ-dependent mutagenic extension, thus shaping mutational frequencies and signatures. • The signature of adduct-induced mutations in mismatch repair-deficient cells matches human tumors following chemotherapy. [ABSTRACT FROM AUTHOR]

[Display omitted] Carbon black nanoparticles (CBNPs) have been demonstrated to induce DNA damage in epithelial cells. However, the potential of the damage to initiate carcinogenesis and the underlying mechanism remain poorly understood. Therefore, we constructed an in vitro model of malignant transformation of human bronchial epithelial cells (16HBE-T) by treating 40 μg/mL CBNPs for 120 passages. We observed tumor-like transformation and sustained DNA damage. Using transcriptome sequencing and RIP-seq, we identified the overexpression of the critical DNA mismatch repair genes MutS homolog 2 (MSH2) and its related circular RNA, circ_0025373, in the 16HBE-T cells. Mechanistically, circ_0025373 was found to inhibit DNA damage by binding to MSH2, thereby modifying its expression and influencing its nuclear and cytoplasmic distribution, which lead to inhibition of CBNP-induced malignant transformation of human bronchial epithelial cells. Our findings provide novel evidence on the carcinogenicity of CBNPs, and offer biological insights into the potential epigenetic regulation and potential therapeutic targets for lung carcinogenesis. [ABSTRACT FROM AUTHOR]

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 heighthroughput 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. [ABSTRACT FROM AUTHOR]

Breast cancer risk associated with germline likely pathogenic/pathogenic variants (PV) varies by gene, often by penetrance (high >50% or moderate 20–50%), and specific locus. Germline PVs in BRCA1 and BRCA2 play important roles in the development of breast and ovarian cancer in particular, as well as in other cancers such as pancreatic and prostate cancers and melanoma. Recent studies suggest that other cancer susceptibility genes, including ATM, CHEK2, PALB2, RAD51C and RAD51D confer differential risks of breast and other specific cancers. In the era of multigene panel testing, advances in next-generation sequencing technologies have notably reduced costs in the United States (US) and enabled sequencing of BRCA1/2 concomitantly with additional genes. The use of multigene-panel testing is beginning to expand in Europe as well. Further research into the clinical implications of variants in moderate penetrance genes, particularly in unaffected carriers, is needed for appropriate counselling and risk management with data-driven plans for surveillance and/or risk reduction. For individuals at high risk without any pathogenic or likely pathogenic variant in cancer susceptibility genes or some carriers of pathogenic variants in moderate-risk genes such as ATM and CHEK2, polygenic risk scores offer promise to help stratify breast cancer risk and guide appropriate risk management options. Cancer patients whose tumours are driven by the loss of function of both copies of a predisposition gene may benefit from therapies targeting the biological alterations induced by the dysfunctional gene e.g. poly ADP ribose polymerase (PARP) inhibitors and other novel pathway agents in cancers with DNA repair deficiencies. A better understanding of mechanisms by which germline variants drive various malignancies may lead to improvements in both therapeutic and preventive management options. • The interpretation of genetic testing results requires careful attention. • ATM, CHEK2, RAD51D and BARD1 correlated with breast and other cancers risk. • European and American guidelines discrepancies. • Support European healthcare providers in interpreting and managing female carriers. [ABSTRACT FROM AUTHOR]

Recent genome-wide association studies have shown that the majority of genes involved in menopause are also instrumental in double-strand break repair and mismatch and base excision repair of DNA. Cumulative DNA damage causes cellular senescence resulting in exhaustion of somatic cell renewal capacity and cellular dysfunction, and eventually to accelerated cell death, generally called ageing. A similar erosion of the genome occurs within the germ cell line and thus in the ovaries. Subsequently, the systemic 'survival' response intentionally suppresses the sex-steroid hormone output, which in turn may contribute to the onset of menopause. The latter occurs in particular when age-dependent DNA damage accumulates. Both effects are expected to synergize to promote ovarian silencing resulting in menopause. Consequently, ageing of the soma seems to be a primary driver for the loss of ovarian function in women. Therefore menopause is the result rather than the cause of ageing. [ABSTRACT FROM AUTHOR]

We have demonstrated in previous work that the Calculus of Covalent Bonding (CCB) can be used to simulate higher-level biochemical processes. This is significant since CCB was originally devised to model lower-level organic chemical reactions. In this paper we extend the use of the calculus to model an important gene repair pathway, namely DNA Mismatch Repair (MMR). This complex pathway involves four helper proteins and needs a distinction between the two chains in a DNA strand. In order to achieve this, we extend the calculus by allowing prefixing with collections of bonding sites. • Calculus of Covalent Bonding (CCB) is extended with a prefix operator to represent multiple bonding sites of molecules. • Revised operational semantics of CCB allows to model out-of-causal order reversibility. • A faulty DNA strand and four proteins needed to repair it are represented as processes in the extended CCB. • Reactions involved in DNA Mismatch Repair mechanism are modelled as transitions in the extended CCB. [ABSTRACT FROM AUTHOR]

Background: Opioid-induced immunomodulation may be important in colon adenocarcinoma, where tumour DNA mismatch repair (MMR) can determine the level of immune activation with consequences for therapeutic response and prognosis. We evaluated the relationship between intraoperative opioid exposure, MMR subtype, and oncological outcomes after surgery for colon adenocarcinoma.Methods: Intraoperative opioid use (standardised by calculating morphine milligram equivalents) during stage I-III colon adenocarcinoma resection was reviewed retrospectively. Tumours were classified as DNA mismatch repair deficient (dMMR) or proficient (pMMR) by immunohistochemistry. The primary outcome was local tumour recurrence, distant tumour recurrence, or both (multivariable analysis). The exposures of interest were intraoperative analgesia and tumour subtype. Opioid-related gene expression was analysed using The Cancer Genome Atlas Colon Adenocarcinoma transcriptomic data.Results: Clinical and pathological data were analysed from 1157 subjects (median age, 60 [51-70] yr; 49% female) who underwent curative resection for stage I-III colon adenocarcinoma. Higher intraoperative opioid doses were associated with reduced risk of tumour recurrence (hazard ratio=0.92 per 10 morphine milligram equivalents; 95% confidence interval [95% CI], 0.87-0.98; P=0.007), but not with overall survival. In tumours deficient in DNA MMR, tumour recurrence was less likely (HR=0.38; 95% CI, 0.21-0.68; P=0.001), with higher opioid dose associated with eightfold lower recurrence rates. Gene expression related to opioid signalling was different between dMMR and pMMR tumours.Conclusions: Higher intraoperative opioid dose was associated with a lower risk of tumour recurrence after surgery for stage I-III colon adenocarcinoma, but particularly so in tumours in which DNA MMR was deficient. [ABSTRACT FROM AUTHOR]

Infertility is associated to multiple types of different genomic instabilities and is a genetic feature of genomic instability syndromes. While the mismatch repair machinery contributes to the maintenance of genome integrity, surprisingly its potential role in infertility is overlooked. Defects in mismatch repair mechanisms contribute to microsatellite instability and genomic instability syndromes, due to the inability to repair newly replicated DNA. This article reviews the literature to date to elucidate the contribution of microsatellite instability to genomic instability syndromes and infertility. The key findings presented reveal microsatellite instability is poorly researched in genomic instability syndromes and infertility. [ABSTRACT FROM AUTHOR]

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 MutSa, histone mark H3K36me3, and H3K36 trimethyltransferase SETD2, which activates the ATM DNA damage signaling pathway. We found that cells depleted of MutSa or SETD2 accumulate 8-oxoG adducts and fail to trigger H2O2-induced ATM activation. Furthermore, we show that SETD2 physically interacts with both MutSa and ATM, which suggests a role for SETD2 in transducing DNA damage signals from lesion-bound MutSa to ATM. Consistently, MutSa and SETD2 are highly coenriched at oxidative damage sites. The data presented here support a model wherein MutSa, SETD2, ATM, and H3K36me3 constitute a positive feedback loop to help cells cope with oxidative DNA damage. [ABSTRACT FROM AUTHOR]

Objective: We aimed at developing a pilot version of an app (Rosa) that can perform digital conversations with breast or ovarian cancer patients about genetic BRCA testing, using chatbot technology, to identify best practices for future patient-focused chatbots.Methods: We chose a commercial chatbot platform and participatory methodology with a team of patient representatives, IT engineers, genetic counselors and clinical geneticists, within a nationwide collaboration. An iterative approach ensured extensive user and formal usability testing during the development process.Results: The development phase lasted for two years until the pilot version was completed in December 2019. The iteration steps disclosed major challenges in the artificial intelligence (AI)-based matching of user provided questions with predefined information in the database, leading initially to high level of fallback answers. We therefore developed strategies to reduce potential language ambiguities (e.g. BRCA1 vs BRCA2) and overcome dialogue confusion. The first prototype contained a database with 500 predefined questions and 67 corresponding predefined answers, while the final version included 2257 predefined questions and 144 predefined answers. Despite the limited AI functionality of the chatbot, the testing revealed that the users liked the layout and found the chatbot trustworthy and reader friendly.Conclusions: Building a health chatbot is challenging, expensive and time consuming with today's technology. The users had a positive attitude to the chatbot, and would use it in a real life setting, if given to them by health care personnel.Practice Implications: We here present a framework for future health chatbot initiatives. The participatory methodology in combination with an iterative approach ensured that the patient perspective was incorporated at every level of the development process. We strongly recommend this approach in patient-centered health innovations. [ABSTRACT FROM AUTHOR]

Clinicians involved in cancer treatment often utilize somatic tumor sequencing to help tailor chemotherapy and immunotherapy. However, somatic tumor sequencing can also identify patients at risk for germline pathogenic variants causing cancer predisposition syndromes like Lynch syndrome. The extent to which clinicians realize this implication of tumor sequencing is currently unclear. We performed a retrospective chart review of Stanford Health Care patients who had somatic variant(s) in the Lynch syndrome genes or microsatellite instability identified on tumor sequencing to determine the proportion of patients who were referred to genetics. Among 6,556 patients who had tumor testing, 90 (1.37%) had findings compatible with Lynch syndrome. Of the 62 patients who had not already seen genetics, 47/62 (75.8%) were not referred to genetics for germline testing. Additionally, 26/47 (55.3%) of these individuals had a tumor type within the Lynch syndrome spectrum. Of the 10 patients who did elect germline testing after tumor sequencing, 3/10 were positive for Lynch syndrome. Our study highlights the need for specific guidelines to inform clinician referral practices on germline follow-up of somatic tumor testing and demonstrates the importance of continued research on the relationship between somatic tumor variants and germline variants to inform such guidelines. [ABSTRACT FROM AUTHOR]

Meningococcal chemoprophylaxis for people in close contact with patients with invasive meningococcal disease (IMD) is necessary for preventing the spread of Neisseria meningitidis. Ciprofloxacin (CIP) is commonly used to treat IMD. However, CIP-resistant N. meningitidis isolates have rapidly evolved worldwide; therefore, rapid and accurate detection of CIP-resistant N. meningitidis is essential. We developed a mismatch amplification mutation assay for identifying gyrA substitutions T91I and D95Y, associated with reduced CIP susceptibility, using two primer sets to detect these variants. Comparison with gyrA sequencing data showed complete congruency. This method enables reliable detection of CIP-resistant N. meningitidis , thus leading to efficient management and control of IMD infections. [ABSTRACT FROM AUTHOR]

Delineating cell-intrinsic and -extrinsic drivers of hematopoietic stem cell (HSC) self-renewal is critical to improve efforts in ex vivo HSC expansion and to better understand leukemia cell biology. To characterize molecular changes at the proteome level, we applied miniaturized, multiplexed sample preparation protocols in combination with mass spectrometry (MS)-based quantitative proteomics to compare normal and Tet2-deficient HSCs, and data-independent acquisition (DIA)-MS to characterize the extracellular environment of HSCs in vivo and during ex vivo expansion. We show that both the cellular and secreted proteome accurately stratify HSCs based on functional potency and mutational status and identify novel molecular components not captured in transcriptomic analyses. On the pre-leukemia side, we reveal that Tet2-deficient HSCs have altered expression of extracellular matrix (ECM) proteins and that interaction with these proteins in artificial niches affects cellular function. Extracellular proteomic analysis further reveals that Tet2-deficient cells create a microenvironment is pro-inflammatory and pro-thrombotic even in young, asymptomatic animals. In HSC expansion assays, proteomics identifies the requirement for intact DNA repair pathways, specifically mismatch repair proteins, as key components of HSC clones capable of extensive self-renewal compared to unsuccessful expansion cultures. Analysis of the secretome of unsuccessful cultures further identifies mast cell proteins as predictive of failure to expand engraftable HSCs. Collectively these data highlight novel proteins to which transcriptomic studies are blind, and open new opportunities for HSC expansion and preleukemic biology, paving the way for future ex vivo and in vivo modulation of HSC function via manipulation of the cells and their extracellular environment. [ABSTRACT FROM AUTHOR]

Multiple separate repair mechanisms safeguard the genome against various types of DNA damage, and their failure can increase the rate of spontaneous mutagenesis. The malfunction of distinct repair mechanisms leads to genomic instability through different mutagenic processes. For example, defective mismatch repair causes high base substitution rates and microsatellite instability, whereas homologous recombination deficiency is characteristically associated with deletions and chromosome instability. This review presents a comprehensive collection of all mutagenic phenotypes associated with the loss of each DNA repair mechanism, drawing on data from a variety of model organisms and mutagenesis assays, and placing greatest emphasis on systematic analyses of human cancer datasets. We describe the latest theories on the mechanism of each mutagenic process, often explained by reliance on an alternative repair pathway or the error-prone replication of unrepaired, damaged DNA. Aided by the concept of mutational signatures, the genomic phenotypes can be used in cancer diagnosis to identify defective DNA repair pathways. • Defective DNA repair elevates the rates of spontaneous mutagenesis. • Deficiency of each DNA repair pathway results in distinct patterns of mutations. • The mechanisms include error-prone alternative repair or replication of damaged DNA. • The etiology of cancer mutation signatures includes DNA repair deficiencies. [ABSTRACT FROM AUTHOR]

• Under NH 4 Cl stress, shrimp hemocyte ROS rises, intensifying DNA damage, elevating apoptosis, and decreasing THC. • NH 4 Cl may induce ER stress and mitochondrial fission via neurotransmitters in shrimp, leading to elevated ROS. • NH 4 Cl can cause DNA damage in shrimp hemocytes, which can be repaired by BER, NER, MMR and HR pathways. • NH 4 Cl can inhibit autophagy in hemocytes of crustaceans and induce apoptosis. Ammonia-N poses a significant threat to aquatic animals. However, the mechanism of ROS production leading to DNA damage in hemocytes of crustaceans is still unclear. Additionally, the mechanism that cells respond to DNA damage by activating complex signaling networks has not been well studied. Therefore, we exposed shrimp to 0, 2, 10, and 20 mg/L NH 4 Cl for 0, 3, 6, 12, 24, 48, and 72 h, and explored the alterations in endoplasmic reticulum stress and mitochondrial fission, DNA damage, repair, autophagy and apoptosis. The findings revealed that ammonia exposure led to an increase in plasma ammonia content and neurotransmitter content (DA, 5-HT, ACh), and significant changes in gene expression of PLC and Ca2+ levels. The expression of disulfide bond formation-related genes (PDI, ERO1) and mitochondrial fission-related genes (Drp1, FIS1) were significantly increased, and the unfolded protein response was initiated. Simultaneously, ammonia-N exposure leads to an increase in ROS levels in hemocytes, resulting in DNA damage. DNA repair and autophagy were considerably influenced by ammonia-N exposure, as evidenced by changes in DNA repair and autophagy-related genes in hemocytes. Subsequently, apoptosis was induced by ammonia-N exposure, and this activation was associated with a caspase-dependent pathway and caspase-independent pathway, ultimately leading to a decrease in total hemocytes count. Overall, we hypothesized that neurotransmitters in the plasma of shrimp after ammonia-N exposure bind to receptors on hemocytes membrane, causing endoplasmic reticulum stress through the PLC-IP3R-Ca2+ signaling pathway and leading to mitochondrial fission. Consequently, this process resulted in increased ROS levels, hindered DNA repair, suppressed autophagy, and activated apoptosis. These cascading effects ultimately led to a reduction in total hemocytes count. The present study provides a molecular support for the understanding of the detrimental toxicity of ammonia-N exposure to crustaceans. [Display omitted] [ABSTRACT FROM AUTHOR]

DNA mismatch repair (MMR) deficiency and the associated microsatellite instability (MSI) phenotype has become a subject of enormous interest in recent years due to the demonstrated efficacy of immune checkpoint inhibitors (ICI) in advanced tumours. Assessing MSI in patients with gastrointestinal tract (GI) cancers is useful to exclude Lynch syndrome, but also to predict benefit for ICI. Following review of the relevant literature, this review article aims to outline the clinicopathologic spectrum of MSI and mismatch repair deficiency (dMMR) in the GI tract, hepatobiliary system and pancreas and discuss the therapeutic consideration in this disease. • Defects in DNA mismatch repair can be seen in several GI tumours. • Tissue based immunohistochemistry and next generation sequencing are used to detect microsatellite instability. • Evidence to support the use of immune checkpoint inhibitors in GI cancers including in both early and late stage disease is evolving. [ABSTRACT FROM AUTHOR]

Colorectal cancer (CRC) is the most common type of newly diagnosed cancer. Metastatic spread and multifactorial chemoresistance have limited the benefits of current therapies. Hence, it is imperative to identify new therapeutic agents to increase treatment efficacy. One of CRC's most promising immunotherapeutic targets is programmed death-1 (PD-1), a cell surface receptor that regulates immune responses. In this paper, we provide an overview of the therapeutic impact of PD-1 in the treatment of CRC. Cancer cells can exploit the PD-1 pathway by upregulating its programmed death-ligand 1 (PD-L1) ligand to evade immune surveillance. The binding of PD-L1 to PD-1 inhibits T cell function, leading to tumor immune escape. PD-1 inhibitors, such as pembrolizumab and nivolumab, block the PD-1/PD-L1 interaction. Clinical trials evaluating PD-1 inhibitors in advanced CRC have shown promising results. In patients with microsatellite instability-high (MSI-H) or mismatch repair-deficient (dMMR) tumors characterized by high mutation rates and increased immunogenicity, PD-1 blockade has demonstrated remarkable efficacy. As a result, pembrolizumab and nivolumab have received accelerated approval by regulatory authorities for the treatment of MSI-H/dMMR metastatic CRC. Additionally, combination approaches, such as combining PD-1 inhibitors with other immunotherapies or targeted agents, are being explored. Despite the success of PD-1 inhibitors in CRC, challenges still exist. Immune-related adverse events can occur and require close monitoring. In conclusion, PD-1 inhibitors have demonstrated significant therapeutic impact, particularly in patients with MSI-H/dMMR tumors. [ABSTRACT FROM AUTHOR]

[Display omitted] • In developed and developing countries, colorectal cancer is ranked as one of the most serious health issues. • Development of nanotechnology has opened new avenues for efficiently organized nanomaterials. • Nanosensors utilizing different nanoparticles have become increasingly popular in recent years. • This review delves into diverse aspects of CRC, including clinical stages and potential biomarkers. As the third most severe cancer in the world, colorectal cancer (CRC) is of high concern to global health concerns. Aging, unhealthy lifestyles, and genetic factors contribute to CRC development. Early detection and intervention are crucial due to early-stage symptoms' subtle or absent nature. The interplay of genetic and environmental factors has identified potential CRC biomarkers for early detection and prognosis. Nanotechnology offers promising opportunities in CRC screening, detection, and treatment. A targeted nanoparticle enhances the accuracy of diagnosis and treatment effectiveness for CRC. PET, MRI, and fluorescence imaging are nanoparticle-based imaging techniques that have revolutionized cancer detection. Additionally, nanoparticles are used in biosensors, highly sensitive devices, to detect CRC-associated biomarkers, allowing for an early diagnosis. Nanoparticles have made significant contributions to cancer care due to these advancements. Drug delivery systems utilize nanoparticles to deliver anticancer drugs directly to CRC cells, improving drug efficacy and stability. This review examines CRC, covering clinical stages, pathogenic mechanisms, and potential biomarkers. Some CRC biomarkers include mutations in genes (KRAS, BRAF, TP53, APC, NRAS), Carcinoembryonic Antigen (CEA), DNA mismatch repair, CA19-9, Epidermal growth factor receptor (EGFR), microsatellite instability status, MicroRNAs (miRNAs), Interleukin-6 (IL-6), CpG island methylation, and Circulating Tumor Cells (CTCs). These biomarkers play a role in diagnosis, prognosis, and targeted treatment strategies for CRC. The review emphasizes the importance of early diagnosis for successful treatment and improved patient outcomes. It explores recent advancements in utilizing nanotechnology for CRC diagnosis and therapy. Integrating nanomedicine and personalized treatments in CRC management shows promise for enhancing patient care and prognosis. Investing in nanomaterials and nanotechnology will likely increase our ability to develop more effective treatment, prevention, and diagnosis strategies to combat CRC, ultimately reducing its impact. [ABSTRACT FROM AUTHOR]

• UV-aged polystyrene was more conducive to biofilm formation than pristine PS. • Biofilm on UV aged PS had more resistance mutations than that on pristine PS. • Ery enhanced biofilm formation and resistance mutations on PS microplastics. • UV aging and Ery repressed rpoS, mutS , and uvrD expressions in biofilm bacteria. Microplastics (MPs) and antibiotics co-occur widely in the environment and pose combined risk to microbial communities. The present study investigated the effects of erythromycin on biofilm formation and resistance mutation of a model bacterium, E. coli , on the surface of pristine and UV-aged polystyrene (PS) MPs sized 1–2 mm. The properties of UV-aged PS were significantly altered compared to pristine PS, with notable increases in specific surface area, carbonyl index, hydrophilicity, and hydroxyl radical content. Importantly, the adsorption capacity of UV-aged PS towards erythromycin was approximately 8-fold higher than that of pristine PS. Biofilms colonizing on UV-aged PS had a greater cell count (5.6 × 108 CFU mg−1) and a higher frequency of resistance mutation (1.0 × 10−7) than those on pristine PS (1.4 × 108 CFU mg−1 and 1.4 × 10−8, respectively). Moreover, erythromycin at 0.1 and 1.0 mg L −1 significantly (p < 0.05) promoted the formation and resistance mutation of biofilm on both pristine and UV-aged PS. DNA sequencing results confirmed that the biofilm resistance was attributed to point mutations in rpoB segment of the bacterial genome. qPCR results demonstrated that both UV aging and erythromycin repressed the expression levels of a global regulator rpoS in biofilm bacteria, as well as two DNA mismatch repair genes mutS and uvrD , which was likely to contribute to increased resistance mutation frequency. [Display omitted] [ABSTRACT FROM AUTHOR]

Purpose: Radioresistance contributes to poor clinical therapeutic efficacy in most cancers. Emerging evidence shows that aberrant DNA damage repair is involved in radioresistance. This study aimed to elucidate the mechanism for radioresistance and explore the precise treatment to sensitize the radioresistant tumors.Methods and Materials: Real-time polymerase chain reaction and Western blot were used to confirm the differential expression of epithelial cell transforming 2 (ECT2) in irradiation-resistant and sensitive cell lines. Laser microirradiation was used to examine the ribosome DNA (rDNA) damage response of ECT2. Biotin-identification, in vivo, in vitro binding assay, and dot blotting were used to confirm the interaction of ECT2 and PARP1. The xenograft mouse model and cell survival assay were used to assess the irradiation sensitivity with or without PARP1 inhibitor.Results: We found the expression of ECT2 correlates with sensitivity to radiation therapy in both lung cancer and nasopharyngeal carcinoma. We demonstrated that low expression of ECT2 causes radioresistance, mainly by protecting rDNA in nucleoli from persistent irradiation exposure through transcriptional recovery prevention. ECT2 is recruited to the rDNA damage site in an ataxia-telangiectasia-mutated RNA polymerase I dependent manner. The recruited ECT2 interacts with PARP1 and facilitates the disassociation of PARP1 from rDNA in nucleoli. Thus, ECT2 deficiency results in sustained activation of PARP1, which subsequently inhibits nucleolar transcription and results in a low frequency of rDNA exposure under DNA damage. PARP inhibition synergized with irradiation can sensitize radioresistant tumors with low ECT2 expression.Conclusions: Our study provides a potential perspective for the application of PARP inhibitor to sensitize low-ECT2 expressing tumors to radiation therapy. [ABSTRACT FROM AUTHOR]

The DNA mismatch repair (MMR) system is a major DNA repair system that corrects DNA replication errors. In eukaryotes, the MMR system functions via mechanisms both dependent on and independent of exonuclease 1 (EXO1), an enzyme that has multiple roles in DNA metabolism. Although the mechanism of EXO1-dependent MMR is well understood, less is known about EXO1-independent MMR. Here, we provide genetic and biochemical evidence that the DNA2 nuclease/helicase has a role in EXO1-independent MMR. Biochemical reactions reconstituted with purified human proteins demonstrated that the nuclease activity of DNA2 promotes an EXO1-independent MMR reaction via a mismatch excision-independent mechanism that involves DNA polymerase δ. We show that DNA polymerase ε is not able to replace DNA polymerase δ in the DNA2-promoted MMR reaction. Unlike its nuclease activity, the helicase activity of DNA2 is dispensable for the ability of the protein to enhance the MMR reaction. Further examination established that DNA2 acts in the EXO1-independent MMR reaction by increasing the strand-displacement activity of DNA polymerase δ. These data reveal a mechanism for EXO1-independent mismatch repair. [ABSTRACT FROM AUTHOR]