Interaction of Xenopus Cdc2 x cyclin A1 with the origin recognition complex.
The initiation of DNA replication in eukaryotes is regulated in a minimum of at least two ways. First, several proteins, including origin recognition complex (ORC), Cdc6 protein, and the minichromosome maintenance (MCM) protein complex, need to be assembled on chromatin before initiation. Second, cyclin-dependent kinases regulate DNA replication in both a positive and a negative way by inducing the initiation of DNA replication at G(1)/S transition and preventing further rounds of origin firing within the same cell cycle. Here we characterize a link between the two levels. Immunoprecipitation of Xenopus origin recognition complex with anti-XOrc1 or anti-XOrc2 antibodies specifically co-immunoprecipitates a histone H1 kinase activity. The kinase activity is sensitive to several inhibitors of cyclin-dependent kinases including 6-dimethylaminopurine (6-DMAP), olomoucine, and p21(Cip1). This kinase activity also copurifies with ORC over several fractionation steps and was identified as a complex of the Cdc2 catalytic subunit and cyclin A1. Neither Cdk2 nor cyclin E could be detected in ORC immunoprecipitations. Reciprocal immunoprecipitations with anti-Xenopus Cdc2 or anti-Xenopus cyclin A1 antibodies specifically co-precipitate XOrc1 and XOrc2. Our results indicate that Xenopus ORC and Cdc2 x cyclin A1 physically interact and demonstrate a physical link between an active cyclin-dependent kinase and proteins involved in the initiation of DNA replication.
Romanowski
P
P
Wellcome/Cancer Research Campaign Institute, Tennis Court Road, Cambridge CB2 1QR, United Kingdom.
Marr
J
J
Madine
M A
MA
Rowles
A
A
Blow
J J
JJ
Gautier
J
J
Laskey
R A
RA
D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D016203:CDC2 Protein Kinase / Q000378:metabolism*; D019925:Cyclin A / Q000378:metabolism*; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000494:pharmacology; D004261:DNA Replication / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007701:Kinetin; D051738:Origin Recognition Complex; D010766:Phosphorylation; D011233:Precipitin Tests; D011494:Protein Kinases / Q000378:metabolism; D011687:Purines / Q000494:pharmacology; D014981:Xenopus; D029867:Xenopus Proteins
D019925:Cyclin A; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D051738:Origin Recognition Complex; D011687:Purines; C097703:XORC2 protein, Xenopus; C102178:XOrc1 protein, Xenopus; D029867:Xenopus Proteins; C001020:N(6),N(6)-dimethyladenine; C090046:olomoucine; D011494:Protein Kinases; C102333:histone H1 kinase; D016203:CDC2 Protein Kinase; D000225:Adenine; D007701:Kinetin