DNA is continuously damaged by both endogenous and exogenous sources. Different type of lesions can be produced, but double strand breaks (DSB) are one of the most toxic forms of genomic lesions, highly threatening genomic integrity, and cell survival (1). If unrepaired, a single DSB can cause cancer and/or other diseases. Although the DNA damage response involves several pathways to ensure the integrity of a genome, some proteins have pivotal roles throughout these processes. RAD52 has been identified as a protein that participates in, or leads, several DNA repair pathways. Given its importance, interest on RAD52 has been reignited recently (2). Different advances have positioned RAD52 as an essential target for the development of medical treatments against cancer and some types of viruses, and drug-targeting of this protein has been shown to be successful 4). However, techniques used so far to determine its full-length structure have not yielded its complete structure due to intrinsic properties of this protein. Hence, further efforts are needed towards determining the structure of the full-length protein (3). Moreover, solving its structure in complex with its substrate nucleic acids will provide additional information about its mechanism of action in DNA damage. Because of its molecular function, this protein can be used to develop new anticancer drugs able to specifically kill cancerous cells in certain types of breast and ovarian cancers, without affecting healthy cells (4).
The aim of this study is to use cryo-EM to determine the structure of the full-length RAD52 bound to its substrate DNA to reveal new insights about its structure and mechanism of action. We have overexpressed the full-length human RAD52 in E. coli and purified it. Negative staining-electron microscopy (NS-TEM) was used to visualize the purified protein and image data analysis was performed in EMAN2 (5) for generating 2D particle classes to further assess the sample quality. As previously reported, RAD52 was found to form oligomeric rings and the 2D classification confirmed the purity and homogeneity of the sample. Further experiments are being performed to obtain homogeneous RAD52+DNA complexes in order to obtain its full-length atomic-resolution structure.