DNA replication of mitotic chromatin in Xenopus egg extracts.
Prereplication complexes are assembled at eukaryotic origins of DNA replication in the G1 phase of the cell cycle, and they are activated in S phase by cyclin-dependent kinase (Cdk)2/cyclin E and Cdk2/cyclin A. Previous experiments using Xenopus nuclear assembly egg extracts suggested that Cdk1/cyclin A, which is normally active in early mitosis, can replace the function of Cdk2 in driving DNA replication, whereas Cdk1/cyclin B, which functions later in mitosis, cannot. Here, we use a completely soluble replication system derived from Xenopus egg extracts to show that Cdk1/cyclin B also can support DNA replication. The ability of mitotic Cdks to drive DNA replication raises the question of whether DNA replication is possible in mitosis. To address this question, chromatin containing prereplication complexes was driven into mitosis with Cdk1/cyclin B. Strikingly, upon addition of a replication extract, the chromatin underwent a complete round of DNA replication. Replicating mitotic chromosomes became visibly decondensed, and, after DNA replication was complete, they recondensed. Our results indicate that there is extensive overlap in the substrate specificity of the major metazoan Cdk/cyclin complexes and that mitosis is not fundamentally incompatible with DNA replication. The results suggest that origins that fail to initiate DNA replication in S phase might still be able to do so in mitosis.
Prokhorova
Tatyana A
TA
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.
Mowrer
Karen
K
Gilbert
Catherine H
CH
Walter
Johannes C
JC
D000818:Animals; D016203:CDC2 Protein Kinase / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D002843:Chromatin / Q000378:metabolism; D019926:Cyclin B / Q000378:metabolism; D004261:DNA Replication; D005260:Female; D006801:Humans; D008297:Male; D008856:Microscopy, Fluorescence; D008938:Mitosis; D009865:Oocytes / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism; D016196:S Phase; D013094:Spermatozoa / Q000378:metabolism; D013379:Substrate Specificity; D013997:Time Factors; D014981:Xenopus
D002843:Chromatin; D019926:Cyclin B; D011494:Protein Kinases; C102333:histone H1 kinase; D016203:CDC2 Protein Kinase