Determining the role of RAD51AP1: a new gene in DNA repair and genomic stability

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DESCRIPTION (provided by applicant): RAD51AP1 (AP1 - Associated Protein 1) is a widely expressed, vertebrate-specific, novel RAD51-interacting and DNA-binding protein that is up-regulated in many tumor types. No direct evidence linked RAD51AP1 to homologous recombinational DNA repair (HRR), and its function has remained mysterious. Using RNAi, we have now accumulated a large body of evidence to show that RAD51AP1 is required for HRR. In addition, both our cell biological knockdown investigations and biochemical studies, conducted by a collaborator, suggest that RAD51AP1 functions downstream of the recombinational mediators either in synapsis or post-synapsis, two poorly understood steps within the HRR pathway. HRR is critical for the repair of spontaneous double-strand breaks (DSBs) in S-phase and radiation- and chemically-induced DSBs in S/G2, and is indispensible for maintaining genomic stability and restarting stalled replication forks. HRR is important in limiting mutagenesis and cancer, but also is a target in both tumor and preventive therapy. Importantly, HRR is impaired in brca2 cells and the BRCA2 protein, like RAD51AP1, directly interacts with RAD51. This proposal on human RAD51AP1 has five main goals: 1) to test our hypothesis that RAD51AP1 functions at an intermediate stage in HRR (i.e. downstream of the recombinational mediators) (Aims 1 & 2); 2) to determine if lack of RAD51AP1, both in human cells stably depleted for RAD51AP1 and in a rad51ap1 knockout from DT40 cells, mirrors or exacerbates the phenotype of other HRR mutants (e.g. brca2) (Aims 1 & 2); 3) to test if the interaction of RAD51AP1, through its C-terminal domain (CTD), with human RAD51 is required for HRR (Aims 2 & 3); 4) to determine if other regions of RAD51AP1 are important for its RAD51- interaction and its role in HRR, including regions conserved with NUCKS (Nuclear, Kinase Substrate), a RAD51AP1 paralog (Aims 3 & 4); and 5) to test if the functions of NUCKS and RAD51AP1 overlap (Aim 4). To meet these goals the following approaches will be taken: a lentiviral shRNA system will be used to extensively deplete RAD51AP1 in several human cell lines, which will be tested for RAD51 and RAD54 foci formation. In addition, the levels of sister chromatid exchanges and mutagenesis will be assessed. A rad51ap1 knockout in the model DT40 chicken system will be developed and characterized, and used with RAD51AP1- depleted human cells to test for the biological significance of the RAD51AP1-RAD51 interaction and of the RAD51AP1-CTD. NUCKS, a RAD51AP1 paralog that lacks the RAD51AP1-CTD motif, will be tested for its role in DNA repair, and regions in RAD51AP1 that are conserved with NUCKS will be tested to determine if they are important for the proper function of RAD51AP1. A human cell two-hybrid system will be used to examine the RAD51 interaction and to test for other RAD51AP1 interactions. Our proposed experiments should help establish the exact role of RAD51AP1 in HRR, a prerequisite for better understanding the cellular responses to chemo- and radiotherapy.