Abstract
Faithful propagation of eukaryotic chromosomes usually requires that no DNA segment be replicated more than once during one cell cycle. Cyclin-dependent kinases (Cdks) are critical for the re-replication controls that inhibit the activities of components of the pre-replication complexes (pre-RCs) following origin activation. The origin recognition complex (ORC) initiates the assembly of pre-RCs at origins of replication and Cdk phosphorylation of ORC is important for the prevention of re-initiation. Here we show that Drosophila melanogaster ORC (DmORC) is phosphorylated in vivo and is a substrate for Cdks in vitro. Cdk phosphorylation of DmORC subunits DmOrc1p and DmOrc2p inhibits the intrinsic ATPase activity of DmORC without affecting ATP binding to DmOrc1p. Moreover, Cdk phosphorylation inhibits the ATP-dependent DNA-binding activity of DmORC in vitro, thus identifying a novel determinant for DmORC-DNA interaction. DmORC is a substrate for both Cdk2 x cyclin E and Cdk1 x cyclin B in vitro. Such phosphorylation of DmORC by Cdk2 x cyclin E, but not by Cdk1 x cyclin B, requires an "RXL" motif in DmOrc1p. We also identify casein kinase 2 (CK2) as a kinase activity in embryonic extracts targeting DmORC for modification. CK2 phosphorylation does not affect ATP hydrolysis by DmORC but modulates the ATP-dependent DNA-binding activity of DmORC. These results suggest molecular mechanisms by which Cdks may inhibit ORC function as part of re-replication control and show that DmORC activity may be modulated in response to phosphorylation by multiple kinases.
Highlights
Initiation of DNA replication involves the ordered assembly of large protein complexes at origins of replication
We found that DmOrc1p from origin recognition complex (ORC)PPase was the only subunit affected by increasing amounts of Cdk21⁄7cyclin E, demonstrating that rDmORC is phosphorylated on at least some of the same sites in the baculovirus system as are phosphorylated by Cdk21⁄7cyclin E in vitro
The direct molecular mechanisms governing the control of ORC function following Cyclin-dependent kinases (Cdks) phosphorylation has not been previously examined
Summary
Initiation of DNA replication involves the ordered assembly of large protein complexes at origins of replication. In Drosophila imaginal disks, the bulk of Orc persists from late G1 into metaphase at which time most of the protein is abruptly degraded before entry into the G1 phase [33] These and other studies suggest that, while the protein levels and chromatin association of Orc remain constant throughout the cell cycle, Orc is selectively removed following origin activation until the cell cycle. It is not clear how the biochemical or cytological behavior of the bulk population of ORC contributes to DNA replication regulation [34]
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