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Papers In Press, published online ahead of print September 25, 2000
Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115
Corresponding Author: johannes_walter{at}hms.harvard.edu
To investigate how the protein kinase cdc7 stimulates DNA replication in metazoans , a soluble cell-free replication system derived from Xenopus eggs is being used. DNA is incubated in egg cytosol to form pre-replication complexes (pre-RCs) and then in nucleoplasmic extract (NPE) to initiate DNA synthesis. We find that cdc7 is greatly enriched in NPE 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 G1/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.
J. Biol. Chem, 10.1074/jbc.M008107200
Submitted on September 5, 2000
Accepted on September 24, 2000
Evidence for sequential action of cdc7 and cdk2 protein kinases during initiation of DNA replication in Xenopus egg extracts
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