Identification of a preinitiation step in DNA replication that is independent of origin recognition complex and cdc6, but dependent on cdk2.
Before initiation of DNA replication, origin recognition complex (ORC) proteins, cdc6, and minichromosome maintenance (MCM) proteins bind to chromatin sequentially and form preinitiation complexes. Using Xenopus laevis egg extracts, we find that after the formation of these complexes and before initiation of DNA replication, cdc6 is rapidly removed from chromatin, possibly degraded by a cdk2-activated, ubiquitin-dependent proteolytic pathway. If this displacement is inhibited, DNA replication fails to initiate. We also find that after assembly of MCM proteins into preinitiation complexes, removal of the ORC from DNA does not block the subsequent initiation of replication. Importantly, under conditions in which both ORC and cdc6 protein are absent from preinitiation complexes, DNA replication is still dependent on cdk2 activity. Therefore, the final steps in the process leading to initiation of DNA replication during S phase of the cell cycle are independent of ORC and cdc6 proteins, but dependent on cdk2 activity.
Hua
X H
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Biology Department, University of California, San Diego, CA 92093-0347, USA.
Newport
J
J
D000818:Animals; D042846:CDC2-CDC28 Kinases; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002843:Chromatin / Q000378:metabolism; D019925:Cyclin A / Q000378:metabolism; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005260:Female; D008297:Male; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism; D051738:Origin Recognition Complex; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D013094:Spermatozoa / Q000378:metabolism; D029867:Xenopus Proteins; D014982:Xenopus laevis
C066503:CDC6 protein, S cerevisiae; D018797:Cell Cycle Proteins; D002843:Chromatin; D019925:Cyclin A; D004268:DNA-Binding Proteins; D051738:Origin Recognition Complex; D029701:Saccharomyces cerevisiae Proteins; C102178:XOrc1 protein, Xenopus; D029867:Xenopus Proteins; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495891:Cdk2 protein, Xenopus; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases