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 mitoses. Unlike G1 nuclei, intact G2 nuclei do not replicate during incubation 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 replication licensing factor which is believed to enter the nucleus when the nuclear membrane breaks down at mitosis, Here, we show that G2 nuclei will initiate a new round of replication in the absence of nuclear membrane permeabilization, if they are preexposed to protein kinase inhibitors in vivo. Competence to rereplicate is generated within 30 min of drug treatment, well before the scheduled onset of mitosis. This demonstrates that a protein kinase-dependent mechanism is continually active in G2 phase to actively prevent regeneration of replication capacity in mammalian cells. Kinase inhibition in G2 cells causes nuclear accumulation 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 Academic Press, Inc.