DNA ligase activities during base excision repair coordination

项目总结 碱基切除修复(BER)是防止病毒引起的突变和致死后果的关键机制 由内源性活性化学物质或暴露在环境危害中造成的DNA损伤。贝尔 是一个多步骤的途径，需要修复蛋白之间的紧密协调。下游的步骤是 BER途径包括DNA聚合酶(POL)β填充缺口和DNA连接酶(连接酶)随后的缺口封闭 I或IIIα)。这种一步步的协调是由非酶支架蛋白X-射线修复十字编排的 互补1(XRCC1)，在组装修复蛋白中起关键作用。尽管个人的角色 酶的研究很多，多蛋白BER复合体如何在维持修复的同时进行协调 效率仍不明朗。尽管误码率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 如果正常的协调能力中断，就会出现不稳定。例如，已发现的Pollβ基因突变 在许多人类癌症中，这会导致其修复功能的修改，从而削弱误码率效率。同样， 具有缺陷支架作用的XRCC1癌症相关变异体使细胞易于发生基因组不稳定性 和转型。BER通路协调失败可能导致链断裂修复的形成 比最初的DNA损伤更具诱变性或毒性的中间体。我的研究计划将填补 误码率领域的重要知识空白--阐明多蛋白误码率的分子组成 对于准确修复和定义缺陷通路协调的分支是必要的复合体 在DNA连接酶I和III的α活性。我们处于独特的地位来推进这一科学战线，基于我们的 良好的记录和我们的多学科方法。在项目1中，我们在以前大量工作的基础上使用 生化和生物物理方法确定PolDNA、β连接酶I和IIIα的分子机制 执行修复路径协调。我们的研究还将阐明与癌症相关的XRCC1和POLβ BER功能改变的变异体是通路协调缺陷的重要决定因素。在项目中 2、利用X射线结晶学，我们将阐明决定DNA底物和连接酶相互作用的特征 在原子分辨率的最终缺口密封步骤中的准确与突变结果。这个项目将是 用Cryo-EM扩展以确定BER多蛋白质复合体的结构 XRCC1决定了准确的修复途径协调。有了这两个项目，我的实验室将推出一个 我的小组进行的研究的新的和独特的方面，试图更好地理解这种机制 多蛋白修复复合体通过它在误码率过程中进行协调，并回答了几个关键问题 严密的协调对于保持我们基因组DNA的完整性、正常功能和如何变化至关重要 这些功能源于修复途径协调失败，从而导致疾病。

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.