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Site‐specific ORC binding, pre‐replication complex assembly and DNA synthesis at Schizosaccharomyces pombe replication origins
Author(s) -
Kong Daochun,
DePamphilis Melvin L.
Publication year - 2002
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1093/emboj/cdf546
Subject(s) - schizosaccharomyces pombe , biology , origin recognition complex , schizosaccharomyces , minichromosome maintenance , dna replication , control of chromosome duplication , eukaryotic dna replication , origin of replication , pre replication complex , licensing factor , microbiology and biotechnology , dna , genetics , saccharomyces cerevisiae , yeast
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 S.pombe replication origin consisting of four genetically required sites, we show that, in situ as well as in vitro , Orc4 binds strongly to the Δ3 site, weakly to the Δ6 site and not at all to the remaining sequences. In situ , the footprint over Δ3 is extended during G 1 phase, but only when Cdc18 is present and Mcm proteins are bound to chromatin. Moreover, this footprint extends into the adjacent Δ2 site, where leading strand DNA synthesis begins. Therefore, we conclude that ARS3001 consists of a single primary ORC binding site that assembles a pre‐replication complex and initiates DNA synthesis, plus an additional novel origin element (Δ9) that neither binds ORC nor functions as a centromere, but does bind an as yet unidentified protein throughout the cell cycle. Schizosaccharomyces pombe may be an appropriate paradigm for the complex origins found in the metazoa.