The interplay of TIMELESS and PARP1 in DNA replication fork stability

TIMELESS 和 PARP1 在 DNA 复制叉稳定性中的相互作用

• 批准号: 10517699
• 负责人: Hyungjin Kim
• 金额: $ 31.36万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10517699
• 关键词: Acute; Attention; Auxins; Binding; Biochemical Genetics; Cell Line; Cells; Chromosome abnormality; Clinical; Collaborations; Complex; DNA; DNA Maintenance; DNA Repair; DNA Repair Enzymes; DNA biosynthesis; DNA replication fork; DNA-Directed DNA Polymerase; Data; Defect; Development; Event; Foundations; Genome; Genomic Instability; Genotoxic Stress; Human; Human Cell Line; Human Engineering; Impairment; Knowledge; Link; Maintenance; Malignant Neoplasms; Mediating; Molecular; Movement; Names; Nature; Okazaki fragments; Pathologic; Pathway interactions; Poly Adenosine Diphosphate Ribose; Poly(ADP-ribose) Polymerases; Polymerase; Process; Proteins; Proteolysis; Regulation; Replication-Associated Process; Role; Scheme; Signal Transduction; Single-Stranded DNA; Source; Specific qualifier value; Stress; Stress Response Signaling; Structure; System; Therapeutic Intervention; Transact; Ubiquitin; Work; base; biological adaptation to stress; brca gene; genetic approach; helicase; human disease; insight; neoplastic cell; novel; preservation; prevent; replication stress; scaffold; targeted cancer therapy; targeted treatment; treatment strategy; tumorigenesis

PROJECT SUMMARY DNA is particularly vulnerable to damage during replication, and perturbation of the replisome activity leads to the accumulation of stalled replication forks, causing DNA breakage and chromosomal abnormalities. Such genome instability is aggravated when genome surveillance mechanisms get disrupted at replication forks, which is known to be a key event for tumorigenesis. Hence, knowledge on the principles by which DNA replication fork integrity is preserved is critical for understanding the molecular checkpoint that keeps genome stable and developing strategies for the treatment of human diseases, including cancer where DNA replication goes awry. The DNA replication fork is supported by the multi-protein replication machinery that coordinates DNA unwinding and nascent strand synthesis, which is undertaken by the CMG helicase and DNA polymerases. TIMELESS (TIM) and its heterodimeric partner TIPIN constitute a core scaffold of the fork protection complex (FPC) that links the helicase-polymerase movement, thus preventing uncoupling of their activities, which otherwise would destabilize the replisome structure and impair replication fork progression. While its name implies a critical role in safeguarding DNA replication, the exact nature of the FPC in engaging diverse protective functions coordinated at both active and stalled forks remains largely uncharacterized. Given that active remodeling of stressed forks and collaboration of DNA replication and repair enzymes are necessary for accommodating many DNA-protein transactions that stabilize and recover stalled forks, the FPC is expected to function as a dynamic platform to coordinate DNA replication processes and adapt stress response signaling to rescue damaged forks. We hereby propose to explicate the function of TIM in the FPC in preserving replication fork integrity and protecting stalled forks. Guided by our extensive preliminary data supporting its dynamic interaction with poly(ADP-ribose) polymerase 1 (PARP1), an emerging regulator of DNA replication and a target of cancer therapy, we hypothesize that the intricate interplay of TIM and PARP1 constitutes a central, yet largely unexplored, mechanism to regulate DNA replication and protect stalled forks at multiple levels, which is reinforced to counteract the fork instability of BRCA1/2-deficient cells. Using cellular, biochemical, and genetic approaches in various engineered human cell lines, we will define the roles of the TIM-PARP1 interaction (1) in the replisome function at active forks, focusing on Okazaki fragment processing, (2) in the process of stalled fork protection via regulation of TIM proteolysis, and (3) in conjunction with the BRCA1/2-dependent fork stabilization pathway. Together, our studies will reveal a new regulatory scheme for the maintenance of DNA replication fork integrity through the collaborative action of TIM and PARP1 within the FPC. Ultimately, such knowledge will provide valuable insight into how DNA replication becomes derailed in human pathological conditions, which could be exploited as a potential target for therapeutic intervention.

项目摘要 DNA在复制过程中尤其容易受到损害，并且重新构体活动的扰动导致 停滞的复制叉的积累，导致DNA断裂和染色体异常。这样的 当基因组监测机制在复制叉时被破坏时，基因组不稳定性会加重， 这是肿瘤发生的关键事件。因此，了解DNA的原理知识 保留复制叉的完整性对于理解保持基因组的分子检查点至关重要 稳定而发展的策略用于治疗人类疾病，包括DNA复制的癌症 出了问题。 DNA复制叉由坐标的多蛋白复制机制支持 DNA放松和新生的链合成，由CMG解旋酶和DNA进行 聚合酶。永恒（蒂姆）及其异二聚体伴侣蒂明（Tipin）构成了叉子的核心脚手架 连接解旋酶 - 聚合酶运动的保护配合物（FPC），从而防止其解偶联 活动，否则会破坏重置体结构并损害复制叉进展。 虽然它的名称意味着在保护DNA复制中起着至关重要的作用，但FPC的确切性质 在活跃和失速的叉子上协调的各种保护功能在很大程度上都没有表现。给出 应力叉的积极重塑以及DNA复制和修复酶的协作是 适应许多稳定和恢复叉的DNA-蛋白质交易所必需的，FPC 预计将充当一个动态平台，以协调DNA复制过程并适应应力 响应信号以拯救受损的叉子。我们特此建议在FPC中解释TIM的功能 保留复制叉的完整性并保护停滞的叉子。在我们广泛的初步数据的指导下 支持其与聚（ADP-核糖）聚合酶1（PARP1）的动态相互作用，这是一种新兴调节剂 DNA复制和癌症治疗的靶标，我们假设Tim和Parp1的复杂相互作用 构成了一种中央但未开发的机制，用于调节DNA复制并保护失速的叉子 在多个级别上，可以抵消BRCA1/2缺陷细胞的叉稳定性。使用蜂窝 在各种工程人类细胞系中的生化和遗传方法，我们将定义 tim-parp1相互作用（1）在活动叉的重新组合函数中 （2）通过调节Tim蛋白水解的档案保护，（3）与 BRCA1/2依赖性叉稳定途径。我们的研究一起将揭示一个新的监管计划 通过Tim和Parp1在 FPC。最终，此类知识将为DNA复制如何脱轨提供宝贵的见解 人类病理状况，可以被利用为治疗干预的潜在靶标。