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J Biol Chem. 2000 Dec 15;275(50):39773-8.

Evidence for sequential action of cdc7 and cdk2 protein kinases during initiation of DNA replication in Xenopus egg extracts.

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  • 1Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA. johannes_walter@hms.harvard.edu

Abstract

To investigate how the protein kinase cdc7 stimulates DNA replication in metazoans, a soluble cell-free replication system derived from Xenopus eggs was used. DNA was incubated in egg cytosol to form prereplication complexes and then in nucleoplasmic extract to initiate DNA synthesis. We find that cdc7 is greatly enriched in nucleoplasmic extract and that this high concentration is essential for efficient DNA replication, supporting previous models that the nucleus activates replication indirectly by sequestering essential components. cdc7 binds to chromatin at the G(1)/S transition before initiation occurs, and it dissociates from chromatin as S phase progresses. The chromatin association of cdc7 requires chromatin-bound MCM. In turn, cdc7 is required to load the initiation factor cdc45 onto the DNA. Finally, efficient replication is observed when chromatin is exposed first to cdc7 and then to cdk2 but not when it is exposed to cdk2 before cdc7. Therefore, the cdc7- and cdk2-dependent initiation steps can be separated, indicating the existence of a novel, stable initiation intermediate. Moreover, the data suggest that cdk2 can only act after cdc7 has executed its function.

PMID:
11005825
[PubMed - indexed for MEDLINE]
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