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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要碱基切除修复（BER）是防止突变和致命后果的关键机制 DNA 损伤是一个多步骤的途径，需要修复蛋白之间的紧密协调。 BER 途径的下游步骤涉及 DNA 聚合酶 (pol) β 的间隙填充以及随后的切口密封通过 DNA 连接酶（LIGI 和 LIGIIIα）这种逐步协调是由非酶支架精心安排的。蛋白质 X 射线修复交叉互补 1 (XRCC1) 在组装修复蛋白中发挥关键作用。尽管对各个酶的作用进行了大量研究，但多蛋白 BER 复合体如何协调虽然保持修复效率仍不清楚，但 BER 通常被认为是一个准确的过程。如果正常协调被破坏，BER 通路协调失败会导致基因组不稳定。可能导致形成比初始链断裂修复中间体更具诱变性或毒性的中间体 DNA 损伤。我们在 DNA 连接酶活动过程中遇到了不准确的 BER 通路协调。在修复反应下游步骤的多蛋白复合物内，导致基因组我们的母提案将提供第一个生化和结构。我们将通过以下内容来阐述主要假设。项目：在项目1中，使用生物化学和生物物理方法，我们将通过以下方式定义分子机制：我们的研究还将由 polβ 和 DNA 连接酶（LIGI 和 LIGIIIα）执行修复途径协调。阐明 XRCC1 和癌症相关 polβ 变体的支架功能缺陷是否会改变在项目 2 中，使用 X 射线，BER 函数可能是通路协调缺陷的决定因素。晶体学和冷冻电镜，我们将阐明 DNA 底物和连接酶相互作用的特征，这些特征决定了在原子分辨率的最终切口密封步骤中准确与诱变结果并研究大由 XRCC1 支架的 BER 多蛋白复合物，以获得对结构体系的新见解修复路径协调。此设备补充要求购买 Nikon Ti2-E。配备全自动 4 色 H-TIRF 模块以及落射荧光模块的显微镜模块，以可视化 BER 协调并测量单分子水平的 DNA 连接酶活性。此次购买将直接支持我们母提案项目 1 和 2 中的实验。使用荧光标记修复蛋白的分子方法与整体高度互补我们目前正在进行的实验将定义 BER 通路协调的动态并提供更多信息。 XRCC1 支架的 polβ/连接酶相互作用的全面视图比单独任何一种方法都给出，从而加强我们项目的影响，了解 DNA 损伤是如何协调的。在多蛋白复合物中修复，并且有缺陷的通路协调的后果将识别新的这些步骤可作为未来合理药物设计的目标，以增强人类健康。

项目成果

期刊论文数量（3）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Molecular editing of NSC-666719 enabling discovery of benzodithiazinedioxide-guanidines as anticancer agents.

NSC-666719 的分子编辑使得发现苯并二噻嗪二氧化胍作为抗癌药物成为可能。

DOI：
发表时间：
2024-03-20
期刊：
影响因子：
4.1
作者：
Krishna Rao, Vajja;Paul, Subarno;Gulkis, Mitchell;Shen, Zhihang;Nair, Haritha;Singh, Amandeep;Li, Chenglong;Sharma, Arun K;Çağlayan, Melike;Das, Chinmay;Das, Biswajit;Kundu, Chanakya N;Narayan, Satya;Guchhait, Sankar K
通讯作者：
Guchhait, Sankar K

Unfilled gaps by polβ lead to aberrant ligation by LIG1 at the downstream steps of base excision repair pathway.

polβ 未填充的间隙导致 LIG1 在碱基切除修复途径的下游步骤异常连接。

DOI：
发表时间：
2024-04-24
期刊：
影响因子：
14.9
作者：
Gulkis, Mitchell;Martinez, Ernesto;Almohdar, Danah;Çağlayan, Melike
通讯作者：
Çağlayan, Melike

Uncovering nick DNA binding by LIG1 at the single-molecule level.

在单分子水平上揭示 LIG1 与切口 DNA 的结合。

DOI：
发表时间：
2024-03-29
期刊：
影响因子：
0
作者：
Chatterjee, Surajit;Chaubet, Loïc;van den Berg, Aafke;Mukhortava, Ann;Gulkis, Mitch;Çağlayan, Melike
通讯作者：
Çağlayan, Melike