Replication protein A1 from L. donovani interacts with RECQb via the C-terminal OB fold and stimulates its helicase activity on branched substrates.

RecQ helicases are ubiquitous genome maintenance proteins that can resolve complex DNA structures during replication, recombination, and repair. Eukaryotic RecQ helicases are known to interact with replication protein A (RPA), leading to the stimulation of their unwinding activity. In higher eukaryotes, this interaction is mediated through the acidic residues of RecQ helicases and the N-terminal OB fold (DBD-F) of RPA70. In kinetoplastid parasites, the RPA70 homolog lacks this N-terminal DBD-F domain, raising the possibility of alternative interaction mechanisms. Here, we investigate the physical and functional interaction between the catalytic core of RECQb helicase and RPA1 from Leishmania donovani parasite. Using molecular docking, GST pull-down assays, and Surface Plasmon Resonance analysis (SPR), we demonstrate that the winged-helix (WH) domain of LdRECQb interacts with the C-terminal OB fold 3 (OBF3) of LdRPA1, unraveling a non-conventional interaction mechanism. Functional assays reveal that LdRPA1 stimulates LdRECQb_heli (catalytic core) unwinding activity on long DNA duplexes, Holliday junctions, and G-quadruplex structures without affecting its ATP hydrolysis. Deletion of the OBF3 drastically reduces but does not abolish this stimulatory effect completely. Our results suggest that while OBF3 predominantly interacts with the LdRECQb, other OB folds of RPA1 may also contribute to this stimulatory effect. Furthermore, we demonstrate that the LdRECQb-LdRPA1 association facilitates restoration of model-regressed replication forks in vitro, suggesting a conserved role in replication stress response. Our results reveal a new C-terminus-mediated RPA-RecQ interaction in L. donovani that resembles the prokaryotic SSB-RecQ interaction mechanism, while maintaining functional conservation of eukaryotic DNA metabolism.