Investigating the role of RAD51AP1 in homologous recombination DNA repair

DNA double-strand breaks (DSBs) are considered the most detrimental DNA lesions induced by ionizing radiation (IR). Cells respond to DSBs by activating DNA damage response pathways that include cell cycle arrest and DNA damage repair. Flawless DNA repair through homologous recombination (HR) is essential for correcting DSBs, maintaining genome integrity, and preventing cancer. A pivotal step in HR involves the joining of homologous DNA strands via formation of a presynaptic RAD51 nucleoprotein filament and subsequent displacement-loop. During this step of the HR reaction, RAD51 interacts with RAD51-Associated Protein 1(RAD51AP1). Our previous work has shown that RAD51AP1 is critical for proper HR and for enhancing the activity of RAD51. Wild-type levels of RAD51AP1 are also essential for protecting human cells from the cytotoxic effects of IR exposure as well as for maintaining DNA replication fork progression and counteracting replication stress. Likely related to its ability to mitigate increased levels of DNA replication stress, RAD51AP1 is found to be up-regulated in many types of cancers, including BRCA1-deficient breast cancers. Significantly, RAD51AP1 is mis-regulated in a variety of tumor types, and Kaplan-Meier plots predict poorer overall survival of breast, ovarian, and lung cancer patients with altered RAD51AP1. Nonetheless, how exactly RAD51AP1 functions during the HR reaction is still incompletely understood. Our main objective is to identify and characterize key attributes of RAD51AP1 in the HR reaction. Here, we have characterized further the DNA-binding properties of recombinant human RAD51AP1. Using the immobilized template assay, we find that RAD51AP1 avidly associates with both naked and chromatinized double-stranded (ds)DNA. Deletional and mutational analyses were used to further define the DNA- and chromatin-binding regions in RAD51AP1 via division of the protein into three fragments: F1 (residues 1-94), F2 (residues 95-187), and F3 (residues 188-335). We show that the N-terminal 94 residues of RAD51AP1, which are able to associate with naked dsDNA avidly, are devoid of binding to a chromatinized dsDNA substrate. In contrast, the C-terminal 148 residues of RAD51AP1 show affinity for both naked and chromatinized dsDNA. Two post-translational modification sites that lie within its C-terminal DNA-binding region were also evaluated and showed decreased affinity to chromatinized dsDNA. Based on these findings and other results, we propose a model in which RAD51AP1 guides homology search and hetero-duplex formation after presynaptic filament formation in the HR reaction. By understanding the biology of this important HR protein, we expect to have direct clinical relevance with knowledge that may lead to the advancement and optimization of targeted cancer therapies. Citation Format: Elena Pires, Neelam Sharma, Claudia Wiese, Patrick Sung, Weixing Zhao. Investigating the role of RAD51AP1 in homologous recombination DNA repair [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1749.