A large C-terminal Rad52 segment acts as a chaperone to Form and Stabilize Rad51 Filaments

Introduction

Homologous recombination (HR) is an important pathway for DNA double-strand break (DSB) repair that exists in all kingdoms of life¹. HR also contributes to the robustness of DNA replication through multiple mechanisms, including stabilizing and protecting replication forks^2,3. However, HR is a double-edged sword: it is essential for maintaining genome stability and generating genetic diversity, but it can also lead to genome instability and cell death^4,5. In particular, excessive or unscheduled HR can generate highly toxic, unprocessed HR intermediates^{6, 7–8}. In eukaryotes, the recombinase Rad51 plays a central role in HR by forming nucleoprotein filaments on single-stranded DNA (ssDNA) generated by nucleases from DSBs or from stalled DNA replication forks^{9, 10, 11, 12, 13, 14–15}. These filaments scan the genome for homologous DNA and then perform synapsis and strand invasion, enabling faithful DNA synthesis and repair^{16, 17, 18, 19–20}. Although Rad51 filament formation has been extensively studied, it is still unclear how the regulation of its formation/stability ensures safe HR. Positive regulators of Rad51 filaments support their formation and stability, whereas negative regulators favor their dissociation, limit their extension, and suppress their potential toxicity²¹.

A key activity of positive regulators of Rad51 filaments is to displace RPA (Replication Protein A), which binds to ssDNA at the beginning of HR, to facilitate Rad51 filaments formation (also referenced as the mediator function of positive regulators). In yeast, this activity is mainly carried out by the Rad52 protein, whereas in mammals it is performed by BRCA2^{22, 23, 24, 25, 26, 27, 28, 29–30}. Notably, yeast Rad52 includes an annealase domain that, while absent in BRCA2 is conserved in mammalian RAD52^31,32. In yeast and mammals, Rad51 paralogs are also involved in this process^{33, 34, 35, 36, 37, 38, 39–40}. Rad52 was primarily viewed as a mediator promoting the replacement of RPA by Rad51. There is now increasing evidence for additional roles of Rad52 in Rad51 filament formation and stability. In particular, we have gathered evidence suggesting that Rad52 might also protect Rad51 filaments from dissociation by the Srs2 DNA translocase⁴¹.

In Saccharomyces cerevisiae, Rad52 plays one of the most prominent roles in HR, as evidenced...