Interaction of Xenopus Cdc2 center dot cyclin A1 with the origin recognition complex

The initiation of DNA replication in eukaryotes is regulated in a minimum o f at least two ways. First, several proteins, including origin recognition complex (ORC), Cdc6 protein, and the minichromosome maintenance (MCM) prote in complex, need to be assembled on chromatin before initiation. Second, cy clin-dependent kinases regulate DNA replication in both a positive and a ne gative way by inducing the initiation of DNA replication at G(1)/S transiti on and preventing further rounds of origin firing within the same cell cycl e. Here we characterize a link between the two levels. Immunoprecipitation of Xenopus origin recognition complex with anti XOrc1 or anti-XOrc2 antibod ies specifically co immunoprecipitates a histone H1 kinase activity. The ki nase activity is sensitive to several inhibitors of cyelin-dependent kinase s 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 Al . Neither Cdk2 nor cyclin E could be detected in ORC immunoprecipitations. Reciprocal immunoprecipitations with anti-Xenopus Cdc2 or anti-Xenopus cycl in Al antibodies specifically eo-precipitate XOrc1 and XOrc2. Our results i ndicate that Xenopus ORC and Cdc2.cyclin Al physically interact and demonst rate a physical link between an active cyclin-dependent kinase and proteins involved in the initiation of DNA replication.