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 环境基因毒素，例如氧化剂，烷基化剂，芳香胺，交联药物， 多环芳烃和天然毒素会诱导全部DNA病变，包括无碱性病变 位点，链间交联和笨重的DNA碱加合物。这些环境基因毒素在我们的 水道，食品，工业和农业化学品以及空气污染，并有可能诱发 诱变和基因组不稳定性如果未修复遗传病变。诱变和基因组不稳定性可以 导致发育障碍，衰老和癌症。 HMCES是一种复制耦合的修复蛋白 响应单链DNA无asic位点，并防止其通过AP-Endonecleases进行裂解。卑鄙的遗址是 环境基因毒素引起的常见病变。我的初步结果表明，HMCE可以防止 基因组不稳定性和诱变，我假设它促进了更忠实的修复机制 例如模板切换或叉子逆转。对于本提案的K99阶段，我将学会表现出色，并且 长期诱变测定和DNA深度测序方法，以详细了解HMCE 防止人类细胞中的诱变和基因组不稳定性，并促进更多无错误修复。这项工作将 为研究（R00）创建技术基础和蓝图，以特定于链特异性复制应力 反应以及链特异性障碍和环境基因毒素如何有助于领先和滞后 链诱变。在领先链和滞后链上的复制之间存在核心差异。脱氧核糖核酸 复制不断发生在领先的链上，并在滞后链上不连续。 谴责事件。我假设链特异性病变和障碍会产生差异复制 压力反应和增强诱变方式不同。我将表征滞后和领先的压力 使用公正的方法做出响应。此外，我将确定链特异性应力的后果和 使用基于深序的诱变测定法，基因毒素在诱变链偏置上。最终，这个 提案将推进环境毒理学和DNA维修场，导致范式转移 发现。