Abstract 2394: Rad52 residues associated with colonic adenocarcinoma dictate its cooperative assembly on ssDNA during homologous recombination

DNA repair is essential for maintaining genomic integrity. Homologous recombination (HR) repairs double- stranded DNA breaks (DSBs) and defects lead to the accumulation of mutations, which results in genomic instability and a plethora of breast, ovarian, colonic and skin cancers. HR is initiated when a DSB is recognized and resected by exonucleases to yield single-stranded DNA (ssDNA), which is then coated and protected by replication protein A (RPA). Next, Rad51 recombinases access the ssDNA preoccupied by RPA. The Rad51 filament is then able to undergo strand-invasion and drive the subsequent stages of HR. The four DNA Binding domains in RPA (DBDs A, B, C and D) enable very tight RPA-ssDNA binding. Therefore, exchange of RPA for Rad51 is assisted by a group of proteins called mediators during this pre-synaptic phase of HR. RAD52 is one such pro-recombinogenic mediator and functions to promote RPA-Rad51 exchange. However, the mechanistic details underlying how Rad52 mediates RPA-Rad51 exchange on ssDNA are not fully resolved. Mutations in mediator proteins such as RAD52 and BRCA2 are prevalent in many cancers. For example, the COSMIC database shows there have been 139 sampled cases of mutations of Rad52 in solid tumors. To understand how Rad52 fulfills its mediator functions, it is important to first understand how Rad52 associates with DNA, and how this affects RPA-Rad51 exchange on ssDNA. To this extent, we have systematically characterized the DNA binding properties of Saccharomyces cerevisiae Rad52 and investigated the effects of Rad52 on RPA-DNA binding in different DNA contexts. Our results show that. Rad52 exists as a heptamer and binds to ssDNA with a Kd of 40.2 ± 3.3 nM which is similar to RPA binding to ssDNA with a Kd of 29.2 ± 4 nM. Using Protein Induced Fluorescent Enhancement (PIFE), we show that Rad52 binds cooperatively towards the 5′ end of DNA. This cooperativity arises from DNA binding to two distinct sites in Rad52: outer- and inner-binding site. In humans, mutations in the amino acids located in the inner binding site has been found in adenocarcinoma. We have perturbed and characterized a specific inner site mutation, R37A, that is associated with multiple cases of colonic adenocarcinoma, as well as other amino acids in the inner and outer binding sites. This has allowed us to develop a model of dynamic ssDNA-Rad52 binding. Finally, we show that this cooperativity in Rad52 selectively affects the DNA binding of RPA-DBD-D on a 5′ overhang DNA substrate. A detailed mechanistic insight into Rad52-ssDNA binding will aid in a better understanding of HR, which can contribute to the development of small-molecule inhibitor drugs that can be used as effective chemotherapeutic agents against cancers in the future. Citation Format: Emma A. Tillison, Nilisha Pokhrel, Jaigeeth Deveryshetty, Edwin Antony. Rad52 residues associated with colonic adenocarcinoma dictate its cooperative assembly on ssDNA during homologous recombination [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2394.