The replication capacity of intact mammalian nuclei in Xenopus egg extracts declines with quiescence, but the residual DNA synthesis is independent of Xenopus MCM proteins

In eukaryotes, the initiation of DNA synthesis requires the assembly of a p re-replicative complex (pre-RC) at origins of replication. This involves th e sequential binding of ORC (origin-recognition-complex), Cdc6 and MCM prot eins, a process referred to as licensing. After origin firing, the Cdc6 and MCM proteins dissociate from the chromatin, and do not rebind until after the completion of mitosis, thereby restricting replication to a single roun d in each cell cycle. Although nuclei normally become licensed for replicat ion as they enter G(1), the extent to which the license is retained when ce lls enter the quiescent state (G(0)) is controversial. Here we show that th e replication capacity of nuclei from Swiss 3T3 cells, in Xenopus egg extra cts, is not lost abruptly with the onset of quiescence, but instead decline s gradually, The decline in replication capacity, which affects both the nu mber of nuclei induced to replicate and their subsequent rate of DNA synthe sis, is accompanied by a fall in the level of chromatin-bound MCM2, When qu iescent nuclei are incubated in egg extracts, they do not bind further MCMs unless the nuclei are first permeabilized, The residual replication capaci ty of intact nuclei must therefore be dependent on the remaining endogenous MCMs, Although high levels of Cdk activity are known to block MCM binding, we show that the failure of intact nuclei in egg extracts to increase thei r bound MCMs is not due to their uptake and accumulation of Cdk complexes. Instead, the failure of binding must be due to exclusion of some other bind ing factor from the nucleus, or to the presence within nuclei of an inhibit or of binding other than Cdk activity, In contrast to the situation in Xeno pus egg extracts, following serum stimulation of intact quiescent cells, th e level of bound MCMs does increase before the cells reach S phase, without any disruption of the nuclear envelope.