title: ADP-binding to origin recognition complex of Saccharomyces cerevisiae.
abstract: The origin recognition complex (ORC), a possible initiator of chromosomal DNA replication in eukaryotes, binds to ATP through its subunits Orc1p and Orc5p. Orc1p possesses ATPase activity. As for DnaA, the Escherichia coli initiator, the ATP-DnaA complex is active but the ADP-DnaA complex is inactive for DNA replication and, therefore, the ATPase activity of DnaA inactivates the ATP-DnaA complex to suppress the re-initiation of chromosomal DNA replication. We investigated ADP-binding to ORC by a filter-binding assay. The K(d) values for ADP-binding to wild-type ORC and to ORC-1A (ORC containing Orc1p with a defective Walker A motif) were less than 10nM, showing that Orc5p can bind to ADP with a high affinity, similar to ATP. ORC-5A (ORC containing Orc5p with a defective Walker A motif) did not bind to ADP, suggesting that the ADP-Orc1p complex is too unstable to be detected by the filter-binding assay. ADP dissociated more rapidly than ATP from wild-type ORC and ORC-1A. Origin DNA fragments did not stimulate ADP-binding to any type of ORC. In the presence of ADP, ORC could not bind to origin DNA in a sequence-specific manner. Thus, in eukaryotes, the ADP-ORC complex may be unable to initiate chromosomal DNA replication, and in this it resembles the ADP-DnaA complex in prokaryotes. However, overall control may be different. In eukaryotes, the ADP-ORC complex is unstable, suggesting that the ADP-ORC complex might rapidly become an ATP-ORC complex; whereas in prokaryotes, ADP remains bound to DnaA, keeping DnaA inactive, and preventing re-initiation for some periods.
authors.affiliation: Faculty of Pharmaceutical Sciences, Okayama University, Okayama 700-8530, Japan.
authors.forename: Hitomi
authors.initials: H
authors.lastname: Takenaka
authors.forename: Masaki
authors.initials: M
authors.lastname: Makise
authors.forename: Wakako
authors.initials: W
authors.lastname: Kuwae
authors.forename: Naoko
authors.initials: N
authors.lastname: Takahashi
authors.forename: Tomofusa
authors.initials: T
authors.lastname: Tsuchiya
authors.forename: Tohru
authors.initials: T
authors.lastname: Mizushima
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D001665:Binding Sites; D004261:DNA Replication; D004271:DNA, Fungal / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D051738:Origin Recognition Complex; D011485:Protein Binding; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism
chemical_list: D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D051738:Origin Recognition Complex; D029701:Saccharomyces cerevisiae Proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate