Determining the factors that impact single stranded DNA mutagenesis

确定影响单链 DNA 突变的因素

• 批准号: 10713599
• 负责人: Natalie Saini
• 金额: $ 37.75万
• 依托单位: MEDICAL UNIVERSITY OF SOUTH CAROLINA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2028-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10713599
• 关键词: Acetaldehyde; Alkylating Agents; Bypass; Cell Culture Techniques; Cell Cycle; Cells; Complex; DNA Binding; DNA Damage; DNA Repair; DNA damage checkpoint; DNA glycosylase; Data; Genetic; Genomic Instability; Goals; Human; Individual; Knowledge; Laboratories; Malignant Neoplasms; Mutagenesis; Mutagens; Mutation; Outcome; Pathway interactions; Pattern; Polymerase; Positioning Attribute; Predisposition; Preventive measure; Proteins; Reporter; Research; Research Project Grants; Role; SS DNA BP; Scientific Advances and Accomplishments; Single-Stranded DNA; Specificity; Stretching; System; Work; Yeasts; bioinformatics tool; interdisciplinary approach; large datasets; next generation sequencing; prevent; programs; replication factor A; tool

Abstract Single stranded DNA (ssDNA) has been demonstrated to by extremely vulnerable to DNA damage. Cancers often carry long stretches of clustered mutations that likely arose due to damage of ssDNA. While, various studies have demonstrated that certain mutagens preferentially damage ssDNA, the mechanisms that alter mutation specificity due to damage in ssDNA and the pathways that prevent mutagenesis at ssDNA are unknown. The overarching goal of this proposal is to specifically identify the roles of DNA damage checkpoint proteins, translesion polymerases, ssDNA-specific glycosylases and ssDNA binding proteins in altering the mutation patterns obtained upon ssDNA-specific damage. My laboratory is in a unique position to advance this scientific front based on my strong track record in DNA damage and repair, assembled team of collaborators, and multidisciplinary approach. My expertise in using highly sensitive yeast reporter systems, human cell culture techniques and the use of bioinformatics tools to probe large data sets and to analyze next generation sequencing data allow us to develop our research program to understand the pathways modulating ssDNA mutagenesis in yeast and human cells. Previously, I have demonstrated that alkylating agents and acetaldehyde have an ssDNA-specific mutation signature in yeast and in cancers. These mutation signatures provide us with a highly sensitive tool to determine how changes in various DNA repair, damage bypass and damage sensing pathways alter mutagenesis by ssDNA-specific mutagens. Here, we propose to determine 1) How cell cycle dependent translesion polymerase expression alters mutation signatures in ssDNA; 2) The role of ssDNA binding Replication Protein A complex in protecting ssDNA from exogenous damage; 3) The role of DNA damage checkpoint activation in modulating the mutation signatures associated with ssDNA damage; 4) Which DNA glycosylases function on ssDNA and alter the mutation signatures due to ssDNA damage; and 5) What are the mutagenic outcomes when translesion polymerases are unable to bypass ssDNA damage. This set of research projects will address a key gap in knowledge in understanding the mechanisms that alter the hypermutability of ssDNA in cells. Our work will enable us to identify and develop better cancer preventative measures for individuals who are prone to increased genome instability and ssDNA formation in their cells.

抽象的 单链 DNA (ssDNA) 已被证明极易受到 DNA 损伤。癌症 通常携带很长一段簇状突变，这些突变可能是由于 ssDNA 损伤而引起的。同时，各种 研究表明，某些诱变剂会优先破坏 ssDNA，这种机制会改变 由于 ssDNA 损伤而产生的突变特异性以及防止 ssDNA 突变的途径是 未知。该提案的首要目标是明确 DNA 损伤检查点的作用 蛋白质、跨损伤聚合酶、ssDNA 特异性糖基化酶和 ssDNA 结合蛋白在改变 ssDNA 特异性损伤后获得的突变模式。我的实验室处于推动这一领域的独特地位 科学前沿基于我在 DNA 损伤和修复方面的良好记录，组建了合作者团队， 和多学科方法。我在使用高度敏感的酵母报告系统、人类细胞方面的专业知识 培养技术和使用生物信息学工具来探测大数据集并分析下一代 测序数据使我们能够开发我们的研究计划来了解调节 ssDNA 的途径 酵母和人类细胞中的诱变。之前，我已经证明了烷化剂和 乙醛在酵母和癌症中具有 ssDNA 特异性突变特征。这些突变特征 为我们提供了一个高度敏感的工具来确定各种DNA修复、损伤旁路和修复过程中的变化如何 损伤传感途径通过 ssDNA 特异性诱变剂改变诱变。在这里，我们建议确定1） 细胞周期依赖性跨损伤聚合酶表达如何改变 ssDNA 中的突变特征； 2）角色 ssDNA 结合复制蛋白 A 复合物在保护 ssDNA 免受外源性损伤中的作用； 3）作用 DNA 损伤检查点激活调节与 ssDNA 损伤相关的突变特征； 4） 哪些 DNA 糖基化酶在 ssDNA 上起作用并改变由于 ssDNA 损伤而导致的突变特征；和 5) 当跨损伤聚合酶无法绕过 ssDNA 损伤时，会产生什么诱变结果。这 一系列研究项目将解决在理解改变机制的知识方面的一个关键差距 细胞中 ssDNA 的超突变性。我们的工作将使我们能够识别和开发更好的癌症预防药物 针对那些容易增加基因组不稳定性和细胞内 ssDNA 形成的个体的措施。