The interplay of TIMELESS and PARP1 in DNA replication fork stability

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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.