Purification and characterization of the Schizosaccharomyces pombe origin recognition complex: interaction with origin DNA and Cdc18 protein.
The origin recognition complex (ORC) plays a central role in the initiation of DNA replication in eukaryotic cells. It interacts with origins of DNA replication in chromosomal DNA and recruits additional replication proteins to form functional initiation complexes. These processes have not been well characterized at the biochemical level except in the case of Saccharomyces cerevisiae ORC. We report here the expression, purification, and initial characterization of Schizosaccharomyces pombe ORC (SpORC) containing six recombinant subunits. Purified SpORC binds efficiently to the ars1 origin of DNA replication via the essential Nterminal domain of the SpOrc4 subunit which contains nine AT-hook motifs. Competition binding experiments demonstrated that SpORC binds preferentially to DNA molecules rich in AT-tracts, but does not otherwise exhibit a high degree of sequence specificity. The complex is capable of binding to multiple sites within the ars1 origin of DNA replication with similar affinities, indicating that the sequence requirements for origin recognition in S. pombe are significantly less stringent than in S. cerevisiae. We have also demonstrated that SpORC interacts directly with Cdc18p, an essential fission yeast initiation protein, and recruits it to the ars1 origin in vitro. Recruitment of Cdc18p to chromosomal origins is a likely early step in the initiation of DNA replication in vivo. These data indicate that the purified recombinant SpORC retains at least two of its primary biological functions and that it will be useful for the eventual reconstitution of the initiation reaction with purified proteins.
Chuang
Ray-Yuan
RY
Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Chretien
Louise
L
Dai
Jianli
J
Kelly
Thomas J
TJ
D020816:Amino Acid Motifs; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins; D007313:Insecta; D051738:Origin Recognition Complex; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D012568:Schizosaccharomyces / Q000235:genetics* / Q000378:metabolism*; D029702:Schizosaccharomyces pombe Proteins
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D051738:Origin Recognition Complex; D011994:Recombinant Proteins; D029702:Schizosaccharomyces pombe Proteins; C082462:cdc18 protein, S pombe; D004247:DNA