The mutagenic consequences of replication-coupled DNA repair mechanisms

复制耦合 DNA 修复机制的致突变后果

• 批准号: 10426485
• 负责人: Kavi Mehta
• 金额: $ 10.18万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-22 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10426485
• 关键词: 4-Hydroxy-Tamoxifen; Aging; Agrochemicals; Air Pollution; Alkylating Agents; Aromatic Amines; Aromatic Polycyclic Hydrocarbons; Bacillus subtilis; Binding; Biological Assay; Biological Models; Bypass; CRISPR screen; Cell Nucleus; Cells; Chemicals; Clustered Regularly Interspaced Short Palindromic Repeats; Copy Number Polymorphism; Coupled; Cysteine; Cytidine Deaminase; Cytosine; DNA; DNA Repair; DNA Sequence Rearrangement; DNA biosynthesis; DNA lesion; DNA-(apurinic or apyrimidinic site) lyase; DNA-Directed DNA Polymerase; DNA-protein crosslink; Data; Ensure; Estrogen Receptors; Event; Food; Foundations; Frequencies; Future; Genetic; Genome Stability; Genomic Instability; Genomics; Goals; Human; Hyperactivity; Hypersensitivity; Industrialization; Institution; Lead; Learning; Lesion; Malignant Neoplasms; Mammalian Cell; Methods; Mutagenesis; Mutagens; Mutation; N-terminal; Pathway interactions; Peptide Hydrolases; Pharmaceutical Preparations; Phase; Plasmids; Polymerase; Principal Investigator; Process; Proteins; Research; Research Training; Series; Signal Transduction; Single-Stranded DNA; Site; Source; Specific qualifier value; Specificity; Stress; System; Testing; Toxic Environmental Substances; Toxin; Uracil; Water; Work; adduct; base; biological adaptation to stress; career; crosslink; deep sequencing; developmental disease; endonuclease; environmental toxicology; exome sequencing; inhibitor; innovation; insertion/deletion mutation; lens; multicatalytic endopeptidase complex; mutant; oxidation; prevent; recruit; repaired; replication stress; response; uracil-DNA glycosylase

Project Summary Environmental genotoxins such as oxidation agents, alkylating agents, aromatic amines, crosslinking agents, polycyclic aromatic hydrocarbons, and natural toxins induce a full spectrum of DNA lesions including abasic sites, interstrand crosslinks, and bulky DNA base adducts. These environmental genotoxins are found in our waterways, food, industrial and agricultural chemicals, and air pollution and have the potential to induce mutagenesis and genomic instability if genetic lesions are not repaired. Mutagenesis and genomic instability can lead to developmental disorders, aging, and cancers. HMCES is a replication-coupled repair protein that responds to single-strand DNA abasic sites and prevents their cleavage by AP-endonucleases. Abasic sites are a common lesion caused by environmental genotoxins. My preliminary results suggest that HMCES prevents both genomic instability and mutagenesis, and I hypothesize that it promotes a more faithful repair mechanism such as template switching or fork reversal. For the K99-phase of this proposal I will learn to perform short and long-term mutagenesis assays and DNA deep sequencing methods to understand in detail how HMCES prevents mutagenesis and genomic instability in human cells and promotes more error-free repair. This work will create a technical foundation and blueprint for studies (R00) characterizing the strand-specific replication stress response and how strand-specific obstacles and environmental genotoxins contribute to leading and lagging strand mutagenesis. There are core differences between replication on the leading and lagging strands. DNA replication occurs continuously on the leading strand and discontinuously on the lagging strand through a series of repriming events. I hypothesize that strand-specific lesions and obstacles generate a differential replication stress response, and potentiate mutagenesis differently. I will characterize the lagging and leading strand stress responses using unbiased approaches. Further, I will determine the consequences of strand-specific stress and genotoxins on mutagenic strand bias using deep sequencing-based mutagenesis assays. Ultimately, this proposal will advance the environmental toxicology and DNA repair fields leading to paradigm shifting discoveries.

项目摘要 环境遗传毒素如氧化剂、烷基化剂、芳香胺、交联剂， 多环芳烃和天然毒素诱导全谱DNA损伤，包括脱碱基 位点、链间交联和庞大的DNA碱基加合物。这些环境基因毒素存在于我们的 水、食品、工业和农业化学品以及空气污染， 诱变和基因组不稳定性，如果遗传损伤不修复。突变和基因组不稳定性可以 导致发育障碍、衰老和癌症。HMCES是一种复制偶联修复蛋白， 响应单链DNA脱碱基位点并阻止其被AP内切核酸酶切割。个脱碱基位点 一种由环境遗传毒素引起的常见病变。我的初步结果表明，HMCES可以防止 基因组不稳定性和突变，我假设它促进了一个更忠实的修复机制， 例如模板切换或分叉反转。对于K99阶段的这一建议，我将学习执行短， 长期诱变试验和DNA深度测序方法，以详细了解HMCES 防止人类细胞中的诱变和基因组不稳定性，并促进更无错误的修复。这项工作将 为表征链特异性复制应力的研究（R00）创建技术基础和蓝图 反应以及特定链的障碍和环境遗传毒素如何导致领先和落后 链诱变领先链和滞后链上的复制之间存在核心差异。DNA 复制在前导链上连续发生，在滞后链上不连续地发生， 重新启动事件。我假设链特异性损伤和障碍产生了差异复制 应激反应和不同地加强诱变。我将描述滞后股和领先股的应力 使用无偏的方法。此外，我将确定链特异性应力的后果， 使用基于深度测序的诱变测定，对遗传毒素对诱变链偏倚的影响进行了研究。最终这 该提案将推动环境毒理学和DNA修复领域的发展，导致范式转变 发现。