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: 10797226
• 负责人: MELIKE CAGLAYAN
• 金额: $ 20万
• 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/10797226
• 关键词: Architecture; Base Excision Repairs; Biochemical; Biophysics; Color; Communication; Complement component C1; Cryoelectron Microscopy; DNA; DNA Damage; DNA Ligases; DNA lesion; DNA-Directed DNA Polymerase; Drug Design; Enzymes; Equipment; Failure; Future; Genomic Instability; Health; Human; Individual; Label; Ligase; Malignant Neoplasms; Measures; Microscope; Molecular; Multiprotein Complexes; Outcome; Parents; Pathway interactions; Play; Process; Proteins; Repair Complex; Resolution; Roentgen Rays; Role; Scaffolding Protein; Variant; Visualization; X-Ray Crystallography; biophysical techniques; cytotoxicity; experimental study; insight; novel; prevent; repair function; repaired; response; scaffold; seal; single molecule

PROJECT SUMMARY Base excision repair (BER) is a critical mechanism for preventing the mutagenic and lethal consequences of DNA damage. BER is a 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 (LIGI and LIGIIIα). 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. 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. We hypothesize that inaccurate BER pathway coordination during DNA ligase activities within the multi-protein complex(es) at the downstream steps of the repair response results in genomic instability and cytotoxicity. Our parent proposal will provide the first biochemical and structural characterization of the BER pathway coordination. We will address the main hypothesis with the following projects: In Project 1, using biochemical and biophysical approach, we will define the molecular mechanism by which polβ and DNA ligases (LIGI and LIGIIIα) execute the repair pathway coordination. Our studies will also elucidate whether a defective scaffolding function of XRCC1 and cancer-associated polβ variants with altered BER functions could be determinants of defective pathway coordination. In Project 2, using X-ray crystallography and cryo-EM, 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 and investigate the large BER multi-protein complexes scaffolded by XRCC1 to gain a novel insight into the structural architecture of the repair pathway coordination. This equipment supplement requests the purchase of the Nikon Ti2-E microscope equipped with the fully automated, 4 color H-TIRF module, as well as the epi-fluorescence module, to visualize BER coordination and to measure DNA ligase activities at the single-molecule level. This purchase will directly support the experiments in Projects 1 and 2 of our parent proposal. The single molecule approach using fluorescently labeled repair proteins is highly complementary to the ensemble experiments that we are currently. We will define the dynamics of BER pathway coordination and provide a more comprehensive view of the polβ/ligase interplay scaffolded by XRCC1 than either approach alone would give, thus strengthening the impact of our project. Gaining an understanding of how DNA damage is coordinately repaired within multi-protein complex, and the ramifications of defective pathway coordination will identify novel steps that can be exploited as targets for future rational drug design toward enhancing human health.

项目摘要 碱基切除修复（BER）是防止突变和致命后果的关键机制 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。的 BER途径的下游步骤涉及DNA聚合酶（pol）β的缺口填充和随后的缺口封闭 通过DNA连接酶（LIGI和LIGIIIα）。这种一步一步的协调是由非酶支架精心安排的 X射线修复交叉互补蛋白1（XRCC 1），在组装修复蛋白中起关键作用。 虽然单个酶的作用被大量研究，但多蛋白BER复合物如何协调 同时保持修复效率仍然不清楚。虽然经常被认为是一个准确的过程，BER 如果正常的协调功能被破坏，会导致基因组的不稳定。BER通道协调失败 可能导致链断裂修复中间体的形成，这些中间体比初始的 DNA损伤我们推测，在DNA连接酶活动过程中不准确的BER途径协调 在修复反应的下游步骤的多蛋白复合物内， 不稳定性和细胞毒性。我们的母提案将提供第一个生物化学和结构 BER路径协调的表征。我们将通过以下内容来解决主要假设 项目：在项目1中，使用生物化学和生物物理方法，我们将定义分子机制， 其中polβ和DNA连接酶（LIGI和LIGIIIα）执行修复途径协调。我们的研究也将 阐明XRCC 1和癌症相关polβ变体的支架功能缺陷是否与改变的 BER功能可能是有缺陷的通路协调的决定因素。在项目2中，使用X射线 晶体学和cryo-EM，我们将阐明DNA底物和连接酶相互作用的特点， 在原子分辨率下的最终切口密封步骤中的准确性与致突变性结果，并研究 BER多蛋白质复合物的支架XRCC 1获得一个新的见解的结构架构， 修复途径协调。本设备补充要求购买Nikon Ti 2-E 显微镜配备了全自动，4色H-TIRF模块，以及落射荧光 模块，以可视化BER协调和测量DNA连接酶的活动在单分子水平。 此次购买将直接支持我们母提案中项目1和项目2的实验。单 使用荧光标记的修复蛋白的分子方法与整体是高度互补的 我们目前正在进行的实验。我们将定义BER途径协调的动态，并提供更多 通过XRCC 1构建的polβ/连接酶相互作用的综合视图比单独的任何一种方法都要全面， 从而加强我们项目的影响力。了解DNA损伤是如何协调地 修复内的多蛋白质复合物，并有缺陷的途径协调的分支将确定新的 这些步骤可以作为未来合理药物设计的目标，以增强人类健康。