The mutagenic consequences of replication-coupled DNA repair mechanisms

复制耦合 DNA 修复机制的致突变后果

基本信息

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
项目类别：
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

项目摘要

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.

项目概要环境基因毒素，如氧化剂、烷化剂、芳香胺、交联剂、多环芳烃和天然毒素会诱发全方位的 DNA 损伤，包括脱碱基位点、链间交联和大体积 DNA 碱基加合物。这些环境基因毒素存在于我们的体内水道、食品、工业和农业化学品以及空气污染，并有可能诱发如果遗传损伤未修复，则会发生突变和基因组不稳定。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 无碱基位点并防止其被 AP 核酸内切酶切割。 Abasic 网站有由环境基因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诱变，我假设它促进了更可靠的修复机制例如模板切换或分叉反转。对于本提案的 K99 阶段，我将学习执行简短且长期诱变分析和 DNA 深度测序方法，详细了解 HMCES 防止人类细胞中的突变和基因组不稳定，并促进更多的无差错修复。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链特异性障碍和环境基因毒素如何导致领先和落后链突变。前导链和滞后链上的复制之间存在核心差异。脱氧核糖核酸复制在前导链上连续发生，在滞后链上不连续地发生，通过一系列重新启动事件。我假设链特异性损伤和障碍会产生差异复制应激反应，并以不同方式增强诱变。我将描述滞后链和前导链应力的特征使用公正的方法做出反应。此外，我将确定特定链应力的后果和使用基于深度测序的诱变测定来确定基因毒素对诱变链偏倚的影响。最终，这该提案将推动环境毒理学和 DNA 修复领域的发展，从而导致范式转变发现。