A PROTEIN KINASE-DEPENDENT BLOCK TO REINITIATION OF DNA-REPLICATION IN G2 PHASE IN MAMMALIAN-CELLS

Eukaryotic cells normally replicate their DNA only once between mitose s. Unlike G1 nuclei, intact G2 nuclei do not replicate during incubati on in Xenopus egg extract, However, artificial permeabilization of the nuclear membrane of G2 nuclei allows induction of new initiations by Xenopus egg extract;This is consistent with the action of a replicatio n licensing factor which is believed to enter the nucleus when the nuc lear membrane breaks down at mitosis, Here, we show that G2 nuclei wil l initiate a new round of replication in the absence of nuclear membra ne permeabilization, if they are preexposed to protein kinase inhibito rs in vivo. Competence to rereplicate is generated within 30 min of dr ug treatment, well before the scheduled onset of mitosis. This demonst rates that a protein kinase-dependent mechanism is continually active in G2 phase to actively prevent regeneration of replication capacity i n mammalian cells. Kinase inhibition in G2 cells causes nuclear accumu lation of replication protein A, Rereplication of kinase-inhibited G2 nuclei also depends on factors supplied by Xenopus egg extract, which are distinct from those required for replication licensing. (C) 1996 A cademic Press, Inc.