Regulation of the mitotic DNA damage response

PROJECT SUMMARY/ABSTRACT Overview: DNA double-strand breaks (DSBs) that are present during mitosis can lead to severe genome instability. While cells have robust DNA repair mechanisms in interphase, these pathways, including non- homologous end joining (NHEJ) and homologous recombination (HR), are suppressed in mitosis. Recent discoveries, including my work, revealed that the alternative mutagenic Polymerase θ-mediated end joining (TMEJ) pathway is active during mitosis. However, the regulatory mechanisms determining when and how TMEJ or other repair processes are activated in mitosis remain unclear. My laboratory identified the E3 ubiquitin ligase RNF168 as a key regulator of mitotic DNA repair, which forms the foundation for further exploration of how mitotic DNA damage responses are controlled. RNF168 activity is suppressed in mitosis, but during G1, S, and G2 cell cycle phases RNF168 ubiquitinates histone H2A (ub-H2A) at DSBs to recruit NHEJ and HR machinery. RNF168-generated ub-H2A regulates DNA end resection at DSBs, a process critical to determining whether NHEJ or HR is activated in interphase cells or if DSBs are protected or subject to TMEJ repair in mitosis. I propose a model where ub-H2A acts as a molecular switch from interphase to mitotic DNA repair programs, and that unresolved S/G2 phase resection elicits mitotic TMEJ. Goals: The overall objective of this research is to unravel the molecular mechanisms that regulate the mitotic DNA damage response. To investigate these mechanisms and address key gaps in knowledge, my laboratory will focus on two major project areas: 1) regulation of the switch from interphase to mitotic DNA damage responses, and 2) regulation of DNA end resection during mitosis and the impact on DNA repair pathway activation. To understand how the mitotic DNA damage response program is activated, the goals of project 1 are to: a) reveal how ub-H2A levels and DNA repair activities impact DSB resolution for S/G2 phase breaks that enter mitosis, b) identify the deubiquitinating enzyme responsible for ub-H2A elimination from DSBs at the G2- mitosis transition, and c) determine how chromatin remodeling impacts ub-H2A levels upon mitotic entry. DNA end resection dictates repair pathway selection at interphase and mitotic DSBs. Because ub-H2A is absent during mitosis, the processes regulating resection and repair pathway selection are unclear. The goals of project 2 are to: a) quantify short- and long-range end resection at DSBs generated prior to and during mitosis, b) reveal the relationship between the DSB genomic location, resection distance, and mutagenesis, and c) identify the factors responsible for limiting resection at mitotic DSBs. Vision: The proposed work builds towards a detailed molecular understanding of how mitotic cells process DSBs. By dissecting the regulatory mechanisms governing the DNA damage response during mitosis, this research will advance our understanding of how genome stability is maintained. Insights gained from these studies could inform strategies to mitigate genome instability.