Elucidating RAD52 architecture through an integrated structural biology approach

Human RAD52 (hRAD52) is a 47 kDa protein of 418 amino acids, which plays important functions in many DNA repair mechanisms such as HR and SSA. Recent studies have shown that simultaneous loss of hRAD52 and other DNA repair proteins, such as BRCA1, BRCA2, PALB2, XAB2 or RAD51 paralogs results in synthetic lethal. This evidence highlights hRAD52 as a promising target for drug discover studies aimed at triggering synthetic lethality in BRCA2 deficient cells. hRAD52 consists of two functionally different domains: the highly conserved N-terminal domain (a.a. 1-208), responsible for oligomerization and DNA binding, and the less conserved C-terminal domain (209-418), which contains the nuclear localization signal sequence (NLS), and binding regions for hRAD51 and RPA. Although the interest in hRAD52 has exponentially grown in the last decade, much remains unknown about its structure and mechanism of action. In particular, the absence of structural information for the C-terminal domain of hRAD52 FL hinders a comprehensive understanding of its interactions with partner interactors. To get novel structural insights on this interesting target, Cryo-EM studies were performed on the hRAD52 FL. While the N-terminal region of RAD52 FL is structured in an undecameric ring, as demonstrated by previous X-ray crystallography studies, the C-terminal domain was poorly resolved and largely absent, highlighting that this region is intrinsically disordered. To address the flexibility of the C-terminal domain and gain additional structural insights, we combined AlphaFold2 simulations and SEC-SAXS experiments. These analyses confirmed the extreme flexibility of the RAD52 C-terminal domain, which can be described as an intrinsically disordered region. This observation further draws attention on the role played by this portion of the protein, which may fold only upon partner binding. These results will significantly inform future investigations into RAD52’s mechanism of action and the development of inhibitors, especially within emerging synthetic lethality strategies.