Project 3: PARP: PAR-dependent, phase-transitioned protein assemblies and DNA repair

项目 3：PARP：PAR 依赖性相变蛋白组装和 DNA 修复

• 批准号: 10271094
• 负责人: TANYA T PAULL
• 金额: $ 33.79万
• 依托单位: UNIVERSITY OF CALIF-LAWRENC BERKELEY LAB
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-27 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10271094
• 关键词: Adenosine Diphosphate Ribose; Affect; Alkylation; Biochemistry; Biophysics; Cells; Cellular Structures; Cellular biology; Characteristics; Clinic; Clinical; Complex; DNA; DNA Damage; DNA Double Strand Break; DNA Repair; DNA Repair Enzymes; DNA Repair Pathway; DNA lesion; DNA replication fork; DNA-dependent protein kinase; Disease; Double Strand Break Repair; Drug Design; EXO1 gene; Enzymes; Excision; Gel; Generations; Glycoside Hydrolases; Histones; Human; Hydrolysis; In Vitro; Length; Lesion; Liquid substance; Malignant Neoplasms; Measures; Methods; Modeling; Modification; Monitor; Outcome; Pattern; Phase; Phase Transition; Poly Adenosine Diphosphate Ribose; Poly(ADP-ribose) Polymerases; Polymers; Post-Translational Protein Processing; Production; Proteins; Recruitment Activity; Regulation; Repair Complex; Research; Resolution; Role; Seeds; Site; Structural Biochemistry; Structure; Tertiary Protein Structure; Testing; Therapeutic; Time; Work; base; cofactor; defined contribution; ds-DNA; fluorescence imaging; frontier; in silico; in vitro Assay; inhibitor/antagonist; insight; mutant; poly ADP-ribose glycohydrolase; poly ADPR glycohydrolase inhib; polymerization; polypeptide; recruit; repaired; replication stress; response; single molecule; small molecule; stress tolerance; structural biology; success; therapeutic target; therapy outcome; tool

SUMMARY- PROJECT 3-PARP: PAR-dependent, phase-transitioned protein assemblies and DNA repair Poly-ADP-ribose polymerases (PARPs) utilize NAD+ as a donor for ADP-ribose modification of proteins and for the subsequent additions that generate poly-ADP-ribose (PAR) on these targets. PARP1 and PARP2 are the predominant enzymes in humans that catalyze PARylation in response to DNA damage and are critical targets of cancer therapeutics. Rapid formation of PAR occurs at many different types of DNA lesions and has been shown to be is required for the timely recruitment of DNA repair enzymes. In addition, PAR recruits polypeptides containing low-complexity domains and intrinsically disordered regions. Some of these disordered proteins form phase transitions between soluble, gel, and fibrous states. PAR thus seeds phase condensates at sites of DNA damage to create a specialized compartment for repairing DNA. Despite the fundamental importance of this phenomenon, we know very little about the structural basis for PARP generation of PAR polymers at DNA damage sites or the mechanistic role of PAR-seeded phase transitions in promoting DNA repair. Here we hypothesize that PAR-driven phase separation regulates DNA repair outcomes at diverse DNA lesions and that the length and structure of PAR polymer is critical for tuning these outcomes. We will test this hypothesis by investigating the mechanistic basis of ADP-ribose addition and PAR formation by PARP1/2 in the context of cofactor HPF1 using structural biology, ensemble biochemistry, and single-molecule methods (Aim 1). Structural insights from this effort will produce a working model for PARP1/2 catalytic activity as well as a toolbox of PARP1/2 mutants that generate varying lengths and structures of PAR. As part of this effort we will also develop inhibitors for the primary glycohydrolase that removes DNA damage-induced ADP-ribose modifications (ARH3). We will probe the role of phase transitions in DNA repair by using FUS and other low-complexity proteins to investigate the structural basis of their interactions with PAR-seeded domains of various lengths (Aim 2). Lastly, we will determine the effects of phase transitions on the recruitment and activity of DNA double-strand break repair complexes in vitro, as well as the effects of these transitions on the repair of multiple types of DNA damage in human cells (Aim 3). These efforts join P3 with the other projects in SBDR5 by collaborating to identify effects of lesion-specific PARylation on the repair of alkylation damage, double-strand breaks, and stalled replication forks. In addition, we will determine the composition of PAR-seeded domains in response to DNA damage and how this changes in response to clinically used PARP and glycohydrolase inhibitors. This project will produce structural and mechanistic insights into the dynamic regulation of PAR synthesis and disassembly that occurs in human cells and will determine how the PAR-seeded phase transitions at these sites affect DNA repair and therapeutic outcomes.

摘要 - 项目3-PARP：依赖性相变的蛋白质组件和DNA修复 多-ADP-核糖聚合酶（PARP）利用NAD+作为蛋白质ADP-核糖修饰的供体 随后在这些目标上产生多-ADP-核糖（PAR）的后续添加。 PARP1和PARP2是 人类中主要的酶催化了响应DNA损伤的核心，并且是关键靶标 癌症治疗学。 PAR的快速形成发生在许多不同类型的DNA病变下 及时募集DNA修复酶是必需的。此外，PAR招募多肽 包含低复杂性结构域和本质上无序的区域。这些无序蛋白质中的一些形式 可溶性，凝胶和纤维态之间的相变。 PAR因此在DNA位置的种子相冷凝 损坏以创建用于修复DNA的专门隔间。尽管这是基本的重要性 现象，我们对DNA的PARP生成parp的结构基础知之甚少 损伤部位或分娩相变的机械作用在促进DNA修复中。我们在这里 假设PAR驱动的相分离调节不同DNA病变处的DNA修复结果，并且 聚合物的长度和结构对于调整这些结果至关重要。我们将通过 在PARP1/2中调查ADP-核糖添加的机理基础 使用结构生物学，集合生物化学和单分子方法的辅助因子HPF1（AIM 1）。结构 这项工作的洞察力将为PARP1/2催化活动产生工作模型，以及一个工具箱 PARP1/2突变体产生不同长度和结构的PAR。作为这项努力的一部分，我们还将发展 去除DNA损伤诱导的ADP-核糖修饰（ARH3）的初级糖醇酶的抑制剂。 我们将使用FUS和其他低复合蛋白探测相变在DNA修复中的作用 研究它们与各个长度的par种子域相互作用的结构基础（AIM 2）。最后， 我们将确定相变的影响对DNA双链断裂的募集和活动的影响 在体外修复复合物，以及这些过渡对多种DNA损伤修复的影响 在人类细胞中（AIM 3）。这些努力通过合作确定效果，加入SBDR5中的其他项目 修复烷基化损伤，双链断裂和停滞复制的病变特异性加油 叉子。此外，我们将根据DNA损伤和 这是如何响应临床使用的PARP和糖醇酶抑制剂的响应而变化的。这个项目将产生 对PAR合成和拆卸的动态调节的结构和机械洞察 人类细胞，并将确定这些位点的种子种子过渡如何影响DNA修复和 治疗结果。