Site-specific ORC binding, pre-replication complex assembly and DNA synthesis at Schizosaccharomyces pombe replication origins

摘要: Previous studies have shown that the Schizo saccharomyces pombe Orc4 subunit is solely responsible for in vitro binding of origin recognition complex (ORC) to specific AT-rich sites within S.pombe replication origins. Using ARS3001, a consisting four genetically required sites, we show that, situ as well vitro, binds strongly Δ3 site, weakly Δ6 site and not at all remaining sequences. In situ, footprint over extended during G1 phase, but only when Cdc18 present Mcm proteins are bound chromatin. Moreover, this extends into adjacent Δ2 where leading strand DNA synthesis begins. Therefore, conclude ARS3001 consists single primary ORC assembles pre-replication initiates synthesis, plus an additional novel element (Δ9) neither nor functions centromere, does bind yet unidentified protein throughout cell cycle. Schizosaccharomyces may be appropriate paradigm origins found metazoa.