Dss1 facilitates Rad51 recruitment downstream of BRCA2/Brh2 in response to DNA damage.

Homologous recombination (HR) is a major pathway for repair of DNA double-strand breaks (DSB), recovery of broken replication forks and formation of meiotic crossovers. HR provides a mechanism to precisely repair damaged DNA in a template-dependent process. The defining step in HR is homologous strand exchange directed by the RecA-related recombinase Rad51. BRCA2 and Brh2, the BRCA2 orthologue in Ustilago maydis, enable recombinational repair of DNA by controlling Rad51. In turn, Dss1, a small intrinsically disordered protein that binds to the C-terminal region of BRCA2/Brh2, regulates BRCA2/Brh2. In the present study, we dissect the interdependency of HR proteins for recruitment to DNA-damage induced foci using fluorescence microscopy and genetics. In U. maydis, Brh2 and Dss1 colocalize at DNA damage-induced foci. Dss1 recruitment to foci is dependent on interaction with full-length Brh2 and Dss1-Brh2 interaction is required for resistance to DNA damage. Further, Dss1 is required for Rad51 and Rec2 focus formation. Interestingly, we find that Rad52 is required for Brh2, Rec2 and Dss1 focus formation. In avian DT40 cells, we likewise show that endogenously tagged DSS1 redistributes into subnuclear foci in response to DNA damaging agents. However, DSS1 foci rarely colocalize with BRCA2 foci. Finally, Dss1 focus formation is inhibited by treatment with the proteasome inhibitor MG132, in both U. maydis and DT40 cells, suggesting a role of ubiquitin in homology-dependent repair.