Abstract LB-164: DNA ligase IV modulates the cellular response to DNA replication stress

Abstract

Abstract Correctly repairing DNA damage is crucial to the survival and genomic integrity of cells. Double strand breaks (DSBs) are particularly problematic DNA damage; failure to properly repair DSBs can precede genomic instability leading to cancer. Because of the high proliferative index of cancer cells, replication toxins are successfully used to treat cancer. The two major DNA DSB repair pathways are homologous recombination and nonhomologous end-joining (NHEJ). DNA ligase IV is a central component of NHEJ, performing the final ligation step to reseal the broken DNA ends, in concert with XRCC4. We determined cells lacking the early acting NHEJ protein, and DNA damage response signaling kinase, DNA-PK, restarted DNA replication forks quicker than wild-type following replication stress. The role of other NHEJ proteins in replication fork restart is unclear. Our goal was to investigate whether ligase IV, a protein that appears to function exclusively in NHEJ, is involved in the replication stress response. We tested the hypothesis that ligase IV delays replication restart following exposure to replication toxins. Ligase IV knockdown was confirmed by qPCR and western blotting. siRNA knockdown of ligase IV alone reduced cell viability and it reduced EdU incorporation after replication stress. The opposite effects of DNA-PK and ligase IV on the speed of replication fork restart suggests that NHEJ, per se, does not directly affect replication fork restart. We previously demonstrated that DNA-PK regulates replication fork restart, at least in part, by phosphorylating RPA32, which in turn regulates ATR activation and downstream checkpoint functions. Ligase IV deficiency may influence replication fork restart indirectly, for example, by causing spontaneous DSBs to persist longer, limiting availability of repair factors needed for fork restart (saturation effect). A not mutually exclusive possibility is that fork restart is hindered by persistent DSBs due to checkpoint activation. Understanding how NHEJ proteins impact the replication stress response may provide opportunities for developing more effective cancer treatments. Citation Format: Grace Hooks, Yasmin Anchondo, Neelam Sharma, Jac Nickoloff, Amanda Ashley. DNA ligase IV modulates the cellular response to DNA replication stress. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-164.