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DNA double strand breaks are repaired by the Homologous Recombination (HR) pathway. Mutations in HR factors, such as BRCA1/2 and related genes cause HR deficiency which drives heritable breast and ovarian cancers. In HR deficient cells, mutations in the DNA repair protein, RAD52, result in synthetic lethality. However, how human RAD52 contributes to the viability of BRCA-deficient cells, through DNA pairing or protein-protein interactions, is poorly understood. RAD52 is comprised of equal sized N-terminal (NTD) and C-terminal domains (CTD). The NTD is highly conserved, and responsible for DNA and RNA pairing activities. The CTD is much less conserved and is largely intrinsically disordered, making it a difficult target to study. The CTD contains an interaction region for the major ssDNA-binding protein, RPA. We aim to determine residues important for this interaction, as well as the role of this interaction in genome stability and HR-deficient cell viability. We confirmed that RAD52 interacts with RPA through its CTD by an in vitro pull down, and we show that this interaction is strengthened by the presence of single-strand DNA. Following backbone assignments of RAD52-CTD, we used 1H,15N-HSQC NMR to show that RPA binds to the CTD of RAD52 within the alpha-helix, between residues 251-274, and identified a novel secondary binding region. Using point mutations of residues involved in binding, we show that the QEM motif is important for maintaining the RAD52:RPA interaction. This structural information will help elucidate the mechanisms of an under-characterized interaction in genome stability and BRCA-deficient cell viability using biochemical and cellular techniques.
DiDomenico et al. (Fri,) studied this question.