CDK Phosphorylation Inhibits the DNA-binding and ATP-hydrolysis Activities of the Drosophila Origin Recognition Complex

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 Cdk2⅐cyclin E and Cdk1⅐cyclin B in vitro. Such phosphorylation of DmORC by Cdk2⅐cyclin E, but not by Cdk1⅐cyclin 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.Prevention of re-replication in eukaryotic cells is essential for the maintenance of genomic ploidy, and mechanisms preventing such reinitiation-associated events are needed because unscheduled or uncontrolled DNA replication initiation can lead to genomic instability (1-5). Re-replication control is achieved through (i) the prevention of repeated origin firing during one S phase and (ii) coordination of chromosomal replication with the segregation of the sister chromatids in mitosis. Genetic studies in the fission yeast Schizosaccharomyces pombe first demonstrated that cyclin-dependent kinases (Cdks) 4 play a prominent role in preventing re-replication during S phase and coupling S phase progression to chromosomal segregation in mitosis, by showing that inhibition of Cdk activity in G 2 /M can induce re-entry into S phase in the absence of mitosis (6 -8). Additional studies in the budding yeast, Saccharomyces cerevisiae, substantiated these findings and demonstrated that some Cdk activity is required for one round of replication and that higher levels accumulating in S phase prevent re-initiation. Thus Cdks have both negative and positive roles in the initiation of DNA replication (9). It is now known that in all eukaryotes studied Cdks contribute to the prevention of re-replication through direct inactivation of pre-RC proteins via multiple redundant mechanisms. This redundancy ensures that deregulation of any single mechanism is not sufficient to induce genomic re-replica...

Regulatory evolution in proteins by turnover and lineage-specific changes of cyclin-dependent kinase consensus sites

“…Even when regulation appears conserved, as has been noted in previous studies (22,23), we found that the numbers and positions of CDK consensus sites were not always conserved. A striking example of this is the linker region of ORC1, which contains a strong cluster of CDK consensus matches in all of the animals and most of the fungi (Figs.…”

“…In addition, CDK target proteins often contain multiple CDK consensus sites closely spaced in their primary amino acid sequence; we refer to these as ''clusters.'' Previous studies have noted that, even when clusters of characterized sites are found in orthologous pre-RC components, the individual consensus sites are not always conserved in position or number (22,23). We refer to this as ''turnover'' of sites and suggest that it is consistent with regulation through mechanisms that impose loose constraints on spacing and number of phosphorylation sites (ref.…”

“…1 and 2a). Sites in this region are phosphorylated by CDK in Drosophila (23) and are involved in CDK-regulated localization and degradation of ORC1 in mammalian cells (38,40,41). Despite the persistence of the cluster over long evolutionary distances, examination of the numbers and positions of individual CDK consensus sites (Fig.…”

Origin Remodeling and Opening in Bacteria Rely on Distinct Assembly States of the DnaA Initiator

“…This behavior is reminiscent of ORC, whereby the specific binding of origins is known to regulate initiator ATPase activity (48,57). Given the phylogenetic kinship between cellular initiators, it seems likely Fig.…”

Cdk1-mediated phosphorylation of Cdc7 suppresses DNA re-replication

“…In all cases where specific phosphorylation sites have been identified, multiple phosphorylation events are required to remove the protein from chromatin, suggesting that there is a common mechanism for Cdk1-dependent hyperphosphorylation in the regulation of DNA replication. [4][5][6][7] Dbf4 degradation at the end of mitosis has previously been suggested as the major regulation mechanism in limiting the activity of the DNA replication activator Dbf4/Drf1-dependent kinase (DDK; Cdc7) function to S phase, at least in budding yeast. [8][9][10][11] This proposal was mainly based on the following observation: Although the level of yeast Cdc7 protein is relatively constant throughout the cell cycle, that of Dbf4 fluctuates as it is low in G1 and the post-metaphase/anaphase transition but high during S and G2 phases.…”