Replication-Associated Repair and Replication Fork Maintenance

SBDR计划项目的项目2(复制相关修复和复制分支维护)侧重于复制分支上多条DNA修复途径的整合及其在维持基因组稳定性中的作用。细胞在复制之前投入大量资源来检测和修复DNA损伤，并在存在复制体停滞损伤的情况下保护活跃的复制叉子。这些过程或协调过程的失败可能会导致癌症和衰老。本研究的目的是使用结构和功能相结合的方法来研究与复制相关的协调损伤识别和修复所需的蛋白质-蛋白质和蛋白质-DNA界面。我们提出了四个目标来研究参与切除修复途径的Keystone蛋白质，这些蛋白质在复制体之前或与复制体协同移除损伤，以及参与叉子稳定性的蛋白质。目的1通过结构内切酶结构域的原子分辨研究以及相对非结构的R末端和C末端结构域与配对蛋白RPA、泛素化的增殖细胞核抗原和DNA连接酶I的相互作用，研究XPG在NER、BER和复制相关修复中作用的结构和功能基础。目的2将研究退火解旋酶SMARCAL1与RPA在停滞复制叉处作用的结构和功能基础。由于SMARCAL1是第一个被证明在失速的叉子上维持基因组完整性的退火解旋酶，从机制水平上理解它的功能是至关重要的。目的3通过与FEN-1和XPG、XRCC1/Ligase III和RPA的相互作用，研究NEIL1启动的氧化碱基BER的结构生物学。目标4将集中于PNKP，它具有对单链和双链断裂修复过程至关重要的激酶和磷酸酶活性，也是Neil指导的BER的重要组成部分。我们将研究PNKP磷酸酶结构域底物的结合，从结构上表征PNKP与其磷酸酶活性的特定抑制剂的相互作用，并询问PNKP与XRCC1/Ligase III的相互作用。 拟议的研究是以SBDR2上一次供资期间产生的主要结果和合作为基础的。它们包括与项目1、3、4和6以及与EMB和SCB核心的试验性互动和实质性协同作用。这些研究的预期结果是更详细的蛋白质-蛋白质和蛋白质-DNA复合体参与复制相关修复的分子图像。产生的信息将阐明一种有前景的DNA修复抑制剂的作用模式，并有助于其优化，并将为新的癌症治疗确定新的潜在靶点。

Project 2 (Replication-Associated Repair and Replication Fork Maintenance) of the SBDR Program Project focuses on the integration of multiple DNA repair pathways at replication forks and their roles in the maintenance of genomic stability. Cells devote significant resources to detecting and repairing DNA damage prior to replication and to protecting active replication forks in the presence of replisome-stalling lesions. Failure in these processes or in their coordination can lead to cancer and aging. The goal of this research is to use a combination of structural and functional approaches to investigate the protein-protein and protein- DNA interfaces required for coordinated damage recognition and repair in association with replication. We propose four Aims to examine keystone proteins involved in excision repair pathways that remove damage prior to or in coordination with the replisome, and with proteins involved in fork stability. Aim 1 will investigate the structural and functional basis for roles of XPG in NER, BER, and replication-associated repair by atomic resolution studies of the structured endonuclease domain and by structural characterization of the relatively unstructured R- and C-terminus domains via their interaction with partner proteins RPA, ubiquitinated PCNA, and DNA Ligase I. Aim 2 will investigate the structural and functional basis for the role of the annealing helicase, SMARCAL1, with RPA at stalled replication forks. Since SMARCAL1 is the first annealing helicase demonstrated to act in maintaining genome integrity at stalled forks, it is critically important to understand its function at a mechanistic level. Aim 3 will investigate the structural biology of NEIL1-initiated BER of oxidized bases, through interactions with FEN-1 and XPG, XRCC1/Ligase III, and with RPA. Aim 4 will focus on PNKP, which has kinase and phosphatase activities critical for both single-strand and double-strand break repair processes and which is also an essential component of NEIL-directed BER. We will investigate PNKP phosphatase domain substrate binding, structurally characterize PNKP interaction with a specific inhibitor of its phosphatase activity, and interrogate the interaction of PNKP with XRCC1/Ligase III. The proposed studies are built upon major findings and collaborations generated during the previous funding period in SBDR2. They include experimental interactions and substantial synergy with Projects 1, 3, 4, and 6, as well as with the EMB and SCB Cores. The anticipated outcome of these studies is a much more detailed molecular picture of the protein-protein and protein-DNA complexes involved in replication-associated repair. The information generated will elucidate the mode of action of a promising DNA repair inhibitor and contribute to its optimization, and will define new potential targets for novel cancer therapies.