Abstract 2570: Colonic adenocarcinoma-associated DNA-binding site mutations in Rad52 perturbs its ssDNA binding polarity and function in homologous recombination
Authors: Tillison Emma A. , Pokhrel Nilisha , Antony Edwin 
Published: August 2019

Double strand DNA breaks (DSB) are repaired by the Homologous recombination (HR) pathway. Defective HR leads to accumulation of mutations that result in genomic instability and a plethora of associated cancers including, breast, ovarian, and skin cancers. In HR, the DSB is recognized and resected to yield single-stranded DNA (ssDNA) that are immediately coated by replication protein A (RPA). The Rad51 recombinase, which is the central engine for HR, needs to bind to ssDNA buried under RPA and form a nucleoprotein filament. Cells possess mediator proteins that either promote or inhibit the exchange of RPA for Rad51 on ssDNA. RAD52 is one such pro-recombinogenic mediator and functions analogous to the Breast Cancer type 2 Susceptibility Protein (BRCA2) to promote the formation of the Rad51 filament. However, the mechanistic details underlying how Rad52 mediates RPA-Rad51 exchange on ssDNA remains a mystery. Mutations in mediator proteins such as BRCA2 and RAD52 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 imperative to first understand how Rad52 associates with DNA. The recent crystal structure of human RAD52 (PDB ID) proposed two ssDNA binding site for RAD52: an outer and an inner binding site, where Arg55 is the interface amino acid between the two sites. To systematically characterize how Rad52 associates with ssDNA, we conducted stopped flow experiments with fluorescently labeled ssDNA. Our results show that Rad52 binds cooperatively to the 5′ end of DNA. Additionally, we created mutations in Rad52-ssDNA binding sites to investigate their role in polarity and cooperativity in depth. These residues and adjacent amino acids are hotspots for mutagenesis in cancers. Abolishing the outer binding site results in a loss of cooperative binding and higher order Rad52-ssDNA complex formation. Mutating the inner binding site abolishes ssDNA binding entirely. Additionally, abolishing the interface arginine residue results in non-selective, rapid binding. This exact amino acid has been mutated in two cases of colonic adenocarcinoma. Our result suggests that both sites are required for maintaining cooperativity and polarity of Rad52 binding to ssDNA. A detailed mechanistic insight into Rad52-ssDNA binding will aid in the development of small-molecule inhibitor drugs that can be used as effective chemotherapeutic agents against cancers in the future.

Note: This abstract was not presented at the meeting.

Citation Format: Emma A. Tillison, Nilisha Pokhrel, Edwin Antony. Colonic adenocarcinoma-associated DNA-binding site mutations in Rad52 perturbs its ssDNA binding polarity and function in homologous recombination [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 2570.