Multiple ORC-binding sites are required for efficient MCM loading and origin firing in fission yeast.
In most eukaryotes, replication origins are composed of long chromosome regions, and the exact sequences required for origin recognition complex (ORC) and minichromosome maintenance (MCM) complex association remain elusive. Here, we show that two stretches of adenine/thymine residues are collectively essential for a fission yeast chromosomal origin. Chromatin immunoprecipitation assays revealed that the ORC subunits are located within a 1 kb region of ori2004. Analyses of deletion derivatives of ori2004 showed that adenine stretches are required for ORC binding in vivo. Synergistic interaction between ORC and adenine stretches was observed. On the other hand, MCM subunits were localized preferentially to a region near the initiation site, which is distant from adenine stretches. This association was dependent on adenine stretches and stimulated by a non-adenine element. Our results suggest that association of multiple ORC molecules with a replication origin is required for efficient MCM loading and origin firing in fission yeast.
Takahashi
Tatsuro
T
Department of Biology, Graduate School of Science, Osaka University, 1-1 Machikaneyama-cho, Toyonaka, Osaka 560-0043, Japan.
Ohara
Eri
E
Nishitani
Hideo
H
Masukata
Hisao
H
D000225:Adenine / Q000378:metabolism; D002453:Cell Cycle / Q000235:genetics / Q000502:physiology; D018797:Cell Cycle Proteins / Q000378:metabolism; D002843:Chromatin / Q000276:immunology; D004268:DNA-Binding Proteins / Q000378:metabolism*; D051738:Origin Recognition Complex; D011233:Precipitin Tests; D018741:Replication Origin / Q000235:genetics* / Q000502:physiology; D012568:Schizosaccharomyces / Q000235:genetics* / Q000378:metabolism; D029702:Schizosaccharomyces pombe Proteins
D018797:Cell Cycle Proteins; D002843:Chromatin; D004268:DNA-Binding Proteins; D051738:Origin Recognition Complex; D029702:Schizosaccharomyces pombe Proteins; C082462:cdc18 protein, S pombe; C406818:cdt1 protein, S pombe; D000225:Adenine