The interplay of TIMELESS and PARP1 in DNA replication fork stability

项目摘要 DNA在复制过程中特别容易受到损伤，并且复制体活性的扰动导致 停滞的复制叉的积累，导致DNA断裂和染色体异常。等 当基因组监视机制在复制叉处被破坏时， 这是已知的肿瘤发生的关键事件。因此，关于DNA分子 复制叉的完整性是至关重要的理解分子检查点， 稳定和发展的战略，用于治疗人类疾病，包括癌症，其中DNA复制 出了差错DNA复制叉由多蛋白质复制机制支持， DNA解旋和新生链合成，这是由CMG解旋酶和DNA 聚合酶TIMELESS（TIM）及其异源二聚体伴侣TIPIN构成了叉的核心支架， 保护复合物（FPC），其连接解旋酶-聚合酶运动，从而防止它们的解偶联 活动，否则会使复制体结构不稳定并损害复制叉进展。 虽然它的名字暗示了在保护DNA复制中的关键作用，但FPC在参与DNA复制中的确切性质是不确定的。 在活动和失速叉上协调的各种保护功能在很大程度上仍然没有特征。给定 应激叉的主动重塑以及DNA复制和修复酶的协作， 为了适应许多DNA-蛋白质交易，稳定和恢复停滞的分叉，FPC是必要的。 作为一个动态的平台来协调DNA复制过程和适应压力 响应信号，以拯救受损的叉子。我们在此建议解释TIM在FPC中的功能， 保持复制分叉的完整性并保护停滞的分叉。根据我们大量的初步数据 支持其与聚（ADP-核糖）聚合酶1（PARP1）的动态相互作用，PARP1是一种新兴的 DNA复制和癌症治疗的靶点，我们假设TIM和PARP1的复杂相互作用 构成了一个核心的，但在很大程度上尚未探索的机制，以调节DNA复制和保护停滞的叉 在多个水平，这是加强，以抵消叉不稳定性BRCA1/2缺陷细胞。使用蜂窝， 生物化学和遗传学方法在各种工程人类细胞系中，我们将定义 TIM-PARP1相互作用（1）在活跃叉的复制体功能中，关注冈崎片段加工， (2)在通过调节TIM蛋白水解来保护失速叉的过程中，以及（3）与 BRCA1/2依赖性分叉稳定通路。总之，我们的研究将揭示一个新的监管计划， 通过TIM和PARP1的协同作用维持DNA复制叉的完整性。 FPC。最终，这些知识将提供有价值的见解，以了解DNA复制如何成为脱轨， 人类病理条件，这可以作为一个潜在的目标，用于治疗干预。

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.