Defining synthetic lethal relationships with loss of the homologous recombination factor Rad52

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SUMMARY. The long-term goal of this project is to define factors and pathways that are synthetic lethal with loss of the human Rad52 protein. Rad52 plays essential roles in several homology-driven DNA repair pathways, including single strand annealing, transcription-coupled homologous recombination, and mitotic DNA synthesis (MiDAS). Although Rad52 is not essential, Rad52 loss with disruption of either the breast cancer 1 (BRCA1) or breast cancer 2 (BRCA2) genes is synthetic lethal. Thus, Rad52 is an intriguing potential target for treatment of BRCA-deficient cancers. However, the full breadth of pathways and factors that create a state of Rad52- dependence when compromised are not understood, and the long-term goal of this proposal is to address this gap in knowledge. In preliminary data, I present my CRISPR knock-out screen in Rad52 Knock-out (Rad52KO) cells vs. wild-type (Rad52WT) to identify factors that are synthetic lethal with Rad52 (defined here as loss of fitness). I then present secondary screening that identified three top hits causing increased persistent DNA damage and loss of viability in the Rad52KO vs Rad52WT: ERCC6L/PICH, DHX9 and GLE1. From these data, my overall hypothesis is that a key regulator of Mitosis (PICH) and RNA metabolism factors (DHX9 and GLE1) are synthetic lethal with Rad52 due to dependence on Rad52 to resolve replication stress from diverse sources. Aim 1: To define the synthetic lethal relationship between Rad52 and PICH. Rad52 protects genome stability through roles in MiDAS and suppression of replication stress. PICH mediates resolution of anaphase ultrafine bridges (UFBs), a separate pathway to mitigate replication stress. Aim 1a: I posit that these two pathways are partially redundant in preventing accumulation of genotoxic damage tied to replication stress. Namely, I posit that PICH- UFBs will be elevated in Rad52KO cells, and conversely that depletion of PICH will cause elevated Rad52 recruitment to replication stress in mitotic cells, as well as MiDAS. Aim 1b: I also posit that replication stress that persists until mitosis is the source of persistent DNA damage in cells lacking Rad52 and PICH. I will test this by assaying phosphorylated RPA2 (pRPA), γH2AX, and FANCD2 localization in mitotic cells. Aim 2: To define the synthetic lethal relationship between Rad52 and RNA metabolism factors DHX9 and GLE1. DHX9 and GLE1 have been shown to suppress RNA-DNA hybrids (R-loops). Thus, I hypothesize that R-loop-related replication stress underlies synthetic lethality between these genes and Rad52. I will assay whether depletion of these genes increases R-loops, causes elevated levels of mitotic replication stress (i.e. Rad52 accumulation into foci, MiDAS, PICH-UFBs, and the measures of replication stress described in Aim 1b). In summary, these studies will provide insight into how loss of these genes create a dependence on Rad52-mediated mitigation of replication stress in mitosis (i.e., MiDAS), enhance our understanding of genome maintenance mechanisms, with a long- term goal of identifing tumor-specific vulnerabilities for Rad52 inhibitors.