Re-constitution of pre-RC complex

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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 E. 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 tp suppress the re-initiation of chromosomal DNA replication. In this study, 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 10 nM, showing that Orc5p can bind to ADP with a high affinity, similar to ATP. ORC-SA (ORC containing Orc5p with a defective Walker A motif) did not bind to ADP, suggesting that ADP-Orc1p complex is too unstable to be detected by the filter-binding assay. ADP dissociated more rapidly from wild-type ORC and ORC-1A than ATP did. Origin DNA fragments did not stimulate ADP-binding to any types 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 ADP-ORC complex might rapidly become ATP-ORC complex ; whereas in prokaryotes, ADP remains bound to DnaA, keeping DnaA inactive, and preventing re-initiation for some periods.