ADP-binding to Origin Recognition Complex of Saccharomyces cerevisiae

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 Kd values for ADP-binding to wild-type ORC and to ORC-1A (ORC containing Orc1p with a defective Walker A motif) were less than 10 nM, 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.