Investigation of strand-specific DNA replication stress response mechanisms.

链特异性 DNA 复制应激反应机制的研究。

• 批准号: 10536820
• 负责人: Matthew Cranford
• 金额: $ 6.72万
• 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: 2022-09-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10536820
• 关键词: Biochemical; Biological Assay; Biological Models; Cell Culture Techniques; Cell Extracts; Cells; Cellular Assay; Chemical Agents; DNA; DNA Damage; DNA Repair Pathway; DNA Structure; DNA biosynthesis; DNA lesion; Data; Escherichia coli; Exposure to; Fellowship; Gel; Genetic Materials; Genome; Genome Stability; Genomics; Goals; Grant; In Vitro; Intervention; Investigation; Kinetics; Learning; Lesion; Maintenance; Mammalian Cell; Methods; Modeling; Monitor; Outcome; Parents; Pathway interactions; Plasmids; Proteins; Proteomics; Recombinants; Research; Resolution; Saccharomyces cerevisiae; Site; Specificity; System; Techniques; Training; Work; Xenopus; base; biological adaptation to stress; career; daughter cell; egg; experience; inhibitor; intervention effect; loss of function; reconstitution; recruit; repaired; replication stress; response; stress tolerance; Biochemical; Biological Assay; Biological Models; Cell Culture Techniques; Cell Extracts; Cells; Cellular Assay; Chemical Agents; DNA; DNA Damage; DNA Repair Pathway; DNA Structure; DNA biosynthesis; DNA lesion; Data; Escherichia coli; Exposure to; Fellowship; Gel; Genetic Materials; Genome; Genome Stability; Genomics; Goals; Grant; In Vitro; Intervention; Investigation; Kinetics; Learning; Lesion; Maintenance; Mammalian Cell; Methods; Modeling; Monitor; Outcome; Parents; Pathway interactions; Plasmids; Proteins; Proteomics; Recombinants; Research; Resolution; Saccharomyces cerevisiae; Site; Specificity; System; Techniques; Training; Work; Xenopus; base; biological adaptation to stress; career; daughter cell; egg; experience; inhibitor; intervention effect; loss of function; reconstitution; recruit; repaired; replication stress; response; stress tolerance

Project Summary The genome is continuously exposed to chemical agents which modify DNA bases and damage the DNA structure. These unrepaired DNA lesions can become obstacles during replication. This results in stalling of nascent DNA synthesis and generates replication stress. Since each strand of parental DNA is replicated by distinct mechanisms (leading vs. lagging strand synthesis), DNA lesions encountered on different parental strands will generate unique forms of replication stress. Previous studies using cell culture and simple in vitro replication systems have described translesion synthesis, re-priming, and fork reversal as pathways which function to relieve replication stress. However, these approaches cannot selectively stall leading or lagging strand synthesis nor differentiate the cellular mechanisms that function on each strand. I hypothesize that leading and lagging strand replication stress are relieved by distinct mechanisms. I will differentiate between strand-specific mechanisms using the Xenopus egg extract model replication system. I have successfully constructed plasmid substrates which contain leading and lagging strand-specific lesions. My preliminary data demonstrate that lesions on different strands have different effects on the kinetics of nascent DNA synthesis, suggesting that different forms of replication stress are generated. This proposal will investigate the functional pathways which relieve strand-specific replication stress and evaluate the outcomes of each pathway. In Aim 1, I will use gel- based analyses to monitor the DNA intermediates that are formed and determine how lesions on each strand are eventually overcome. I will also use quantitative proteomics to monitor the proteins that are recruited or lost during replication of leading or lagging strand lesions. Together, these approaches will identify candidate pathways which function on different strands. In Aim 2, I will use a loss-of-function approach to determine how strand-specific pathways are controlled and monitor detrimental replication outcomes when select pathways are inactivated. This will be done by selective inhibition and immunodepletion of candidate proteins from the cell extracts. The findings of this proposal will define the specific cellular pathways which respond to specific forms of replication stress and determine how various mechanisms are employed to faithfully replicate the genome.

项目摘要 基因组不断暴露于修饰DNA碱基并损坏的化学剂 DNA结构。这些未修复的DNA病变在复制过程中可能会成为障碍。这导致 停滞新生DNA合成并产生复制应力。由于父母DNA的每一链 通过不同的机制（领先与滞后链合成）复制，遇到的DNA病变 在不同的父母链上，将产生独特的复制应力。先前使用的研究 细胞培养和简单的体外复制系统已经描述了跨性别的合成，重新提出， 叉子逆转作为功能减轻复制应力的途径。但是，这些方法 无法选择性地停滞不前的链或滞后链合成或区分细胞机制 每个链上的功能。我假设领先和滞后的链复制应力缓解了 通过不同的机制。我将使用爪蟾鸡蛋区分链特异性机制 提取模型复制系统。我已经成功构建了包含的质粒底物 铅和滞后特异性病变。我的初步数据表明，不同的病变 链对新生DNA合成的动力学有不同的影响，表明不同形式 产生复制应力。该提案将调查可减轻的功能途径 链特异性复制应力并评估每种途径的结果。在AIM 1中，我将使用凝胶 - 基于分析以监视形成的DNA中间体，并确定每种病变 链最终被克服。我还将使用定量蛋白质组学监测 在复制领先或滞后病变的复制过程中被招募或丢失。这些方法将在一起 确定在不同链上起作用的候选途径。在AIM 2中，我将使用功能丧失 确定链特异性途径并监测有害复制的方法 当选择途径被灭活时的结果。这将通过选择性抑制和 来自细胞提取物的候选蛋白的免疫部门。该提案的发现将定义 对特定形式的复制应力响应的特定细胞途径，并确定如何 采用各种机制来忠实地复制基因组。