CDK phosphorylation inhibits the DNA-binding and ATP-hydrolysis activities of the Drosophila origin recognition complex.
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.
Remus
Dirk
D
Department of Molecular and Cell Biology, Division of Biochemistry and Molecular Biology, University of California, Berkeley, California 94720-3204, USA.
Blanchette
Marco
M
Rio
Donald C
DC
Botchan
Michael R
MR
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry*; D020816:Amino Acid Motifs; D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism; D049109:Cell Proliferation; D003432:Cross-Linking Reagents / Q000494:pharmacology; D019926:Cyclin B / Q000378:metabolism; D019927:Cyclin E / Q000378:metabolism; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D004247:DNA / Q000737:chemistry*; D004261:DNA Replication; D004331:Drosophila melanogaster; D006868:Hydrolysis; D047468:Immunoprecipitation; D013058:Mass Spectrometry; D008957:Models, Genetic; D051738:Origin Recognition Complex; D010766:Phosphorylation; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism; D011687:Purines / Q000494:pharmacology; D016622:Silver Staining; D013997:Time Factors; D014230:Triazoles / Q000494:pharmacology
C405354:4,5,6,7-tetrabromobenzotriazole; C471843:6-((3-chloro)anilino)-2-(isopropyl-2-hydroxyethylamino)-9-isopropylpurine; D003432:Cross-Linking Reagents; D019926:Cyclin B; D019927:Cyclin E; D051738:Origin Recognition Complex; D011687:Purines; D014230:Triazoles; D000255:Adenosine Triphosphate; D004247:DNA; D011494:Protein Kinases; C102333:histone H1 kinase; D018844:Cyclin-Dependent Kinases; D000251:Adenosine Triphosphatases