Martinez Carranza, MarkelVialle, LeaMadru, ClementCordier, FlorenceTekpinar, Ayten DizkiriciHaouz, AhmedSauguet, Ludovic2025-05-102025-05-1020242041-172310.1038/s41467-024-55365-w2-s2.0-85213685198https://doi.org/10.1038/s41467-024-55365-whttps://hdl.handle.net/20.500.14720/11220England, Patrick/0000-0001-6410-5918; Dizkirici Tekpinar, Ayten/0000-0002-0578-5092; Madru, Clement/0000-0002-5412-8822; Haouz, Ahmed/0000-0003-1196-1635; Legrand, Pierre/0000-0003-2431-2255; Martinez-Carranza, Markel/0000-0003-0192-9762Replication Protein A (RPA) plays a pivotal role in DNA replication by coating and protecting exposed single-stranded DNA, and acting as a molecular hub that recruits additional replication factors. We demonstrate that archaeal RPA hosts a winged-helix domain (WH) that interacts with two key actors of the replisome: the DNA primase (PriSL) and the replicative DNA polymerase (PolD). Using an integrative structural biology approach, combining nuclear magnetic resonance, X-ray crystallography and cryo-electron microscopy, we unveil how RPA interacts with PriSL and PolD through two distinct surfaces of the WH domain: an evolutionarily conserved interface and a novel binding site. Finally, RPA is shown to stimulate the activity of PriSL in a WH-dependent manner. This study provides a molecular understanding of the WH-mediated regulatory activity in central replication factors such as RPA, which regulate genome maintenance in Archaea and Eukaryotes.eninfo:eu-repo/semantics/openAccessArticle151Q1Q139738083WOS:001386373100045