DNA ligase activities during base excision repair coordination

碱基切除修复协调过程中的 DNA 连接酶活性

• 批准号: 10679039
• 负责人: MELIKE CAGLAYAN
• 金额: $ 37.41万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-15 至 2027-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10679039
• 关键词: Architecture; Base Excision Repairs; Biochemical; Cells; Chemicals; Communication; Complement component C1; Cryoelectron Microscopy; DNA; DNA Damage; DNA Ligases; DNA Repair; DNA Repair Gene; DNA lesion; DNA ligase I; DNA-Directed DNA Polymerase; Disease; Environmental Hazards; Enzymes; Exposure to; Failure; Genes; Genomic DNA; Genomic Instability; Human; Impairment; Individual; Knowledge; Laboratories; Ligase; Malignant Neoplasms; Modification; Molecular; Multiprotein Complexes; Mutation; Outcome; Pathway interactions; Play; Positioning Attribute; Process; Proteins; Repair Complex; Research; Resolution; Roentgen Rays; Role; Scaffolding Protein; Scientific Advances and Accomplishments; Site; Variant; Work; X-Ray Crystallography; base; biophysical techniques; interdisciplinary approach; prevent; programs; repair function; repaired; scaffold; seal; stem

PROJECT SUMMARY Base excision repair (BER) is a critical mechanism for preventing the mutagenic and lethal consequences of DNA damage generated by endogenous reactive chemical species or exposure to environmental hazards. BER is multi-step pathway that requires a tight coordination between the repair proteins. The downstream steps of BER pathway involves gap filling by DNA polymerase (pol) β and subsequent nick sealing by DNA ligase (ligase I or IIIα). This step-to-step coordination is orchestrated by non-enzymatic scaffolding protein X-Ray Repair Cross Complementing 1 (XRCC1) that plays a key role in assembling repair proteins. Although the roles of the individual enzymes are largely studied, how the multi-protein BER complex coordinates while maintaining the repair efficiency remains unclear. Though often considered an accurate process, the BER can contribute to genome instability if normal coordination breaks down. For example, the mutations in the polβ gene that have been found in many human cancers result in the modifications in its repair functions that impair BER efficiency. Similarly, XRCC1 cancer-associated variants with a defective scaffolding role predispose the cell to genomic instability and transformation. Failure in the BER pathway coordination could result in the formation of strand-break repair intermediates that are more mutagenic or toxic than the initial DNA lesions. My research program will fill the important gap of knowledge in the BER field by elucidating the molecular components of multi-protein BER complex that are necessary for accurate repair and define the ramifications of defective pathway coordination during DNA ligase I and IIIα activities. We are in a unique position to advance this scientific front based on our strong track record and our multidisciplinary approach. In Project 1, we build off our substantial prior work using biochemical and biophysical approach to define the molecular mechanism by which polβ, DNA ligases I and IIIα execute the repair pathway coordination. Our studies will also elucidate cancer-associated XRCC1 and polβ variants with altered BER functions as an important determinants of defective pathway coordination. In Project 2, using X-ray crystallography, we will elucidate the features of DNA substrate and ligase interaction that dictate accurate versus mutagenic outcomes during final nick sealing step at atomic resolution. This project will be extended with cryo-EM to define the structural architecture of large BER multi-protein complexes scaffolded by XRCC1 that dictates accurate repair pathway coordination. With these 2 Projects, my laboratory will launch a new and unique aspect of the research conducted by my group which seeks to better understand the mechanism by which a multi-protein repair complex coordinate during BER and answer several key questions regarding how a tight coordination is vital for maintaining the integrity of our genomic DNA, functions normally and how altering these functions stemming from a failure in the repair pathway coordination leads to disease.

项目摘要 碱基切除修复（BER）是防止突变和致死性后果的关键机制。 内源性反应性化学物质或暴露于环境危害产生的DNA损伤。ber 是一个多步骤的途径，需要修复蛋白之间的紧密协调。下游步骤 BER途径涉及DNA聚合酶（pol）β的缺口填充和随后的DNA连接酶（ligase）的缺口封闭 I或III α）。这种一步一步的协调是由非酶支架蛋白X射线修复交叉 补体1（XRCC1）在组装修复蛋白中起关键作用。虽然个人的角色 人们大量研究酶，研究多蛋白BER复合物如何在维持修复的同时协调 效率仍然不清楚。尽管BER通常被认为是一个准确的过程，但它可以对基因组做出贡献 如果正常的协调被打破，就会出现不稳定。例如，已经发现的pol β基因的突变 在许多人类癌症中，导致其修复功能的改变，从而损害BER效率。同样地， 具有缺陷支架作用的XRCC1癌症相关变体使细胞易于基因组不稳定 和转变。BER途径协调的失败可能导致链断裂修复的形成 比最初的DNA损伤更具致突变性或毒性的中间体。我的研究计划将填补 通过阐明多蛋白BER的分子组成，填补了BER领域的一个重要知识空白 复杂的是必要的准确修复和定义的分歧，有缺陷的途径协调 在DNA连接酶I和III α活性期间。我们处于一个独特的地位，以推进这一科学前沿的基础上，我们的 良好的业绩记录和多学科方法。在项目1中，我们使用 生物化学和生物物理方法来定义pol β、DNA连接酶I和III α 执行修复路径协调。我们的研究还将阐明癌症相关的XRCC 1和pol β 具有改变的BER功能的变体是有缺陷的途径协调的重要决定因素。在项目 2、利用X射线晶体学，我们将阐明DNA底物和连接酶相互作用的特征， 在原子分辨率下，最终切口密封步骤中的准确性与致突变性结果。该项目将 用cryo-EM扩展，以定义由以下支架支撑的大BER多蛋白质复合物的结构架构： XRCC1指示精确的修复途径协调。有了这两个项目，我的实验室将推出一个 我的团队进行的研究的一个新的和独特的方面，旨在更好地了解机制， 多蛋白修复复合物在BER过程中的协调，并回答了几个关键问题， 一个紧密的协调是至关重要的，以维持我们的基因组DNA的完整性，功能正常，以及如何改变 这些源于修复途径协调失败的功能导致疾病。