The roles of the MCM, ORC, and Cdc6 proteins in determining the replication competence of chromatin in quiescent cells

Most eukaryotic cell types can withdraw from proliferative cell cycles and remain quiescent for extended periods. Intact nuclei isolated from quiescen t murine NIH3T3 cells fail to replicate in vitro when incubated in Xenopus egg extracts, although intact nuclei from proliferating cells replicate wel l. Permeabilization of the nuclear envelope rescues the ability of quiescen t nuclei to replicate in the extract. We show that origin replication compl ex (ORC), minichromosome maintenance (MCM), and Cdc6 proteins are all prese nt in early quiescent cells. Immunodepletion of Cdc6 or the MCM complex fro m Xenopus egg extract inhibits replication of permeable, quiescent, but not proliferating, NIH3T3 nuclei. Immunoblotting results demonstrate that mous e homologues of Mcm2, Mcm5, and Cdc6 are displaced from chromatin in quiesc ent cells. However, this absence of chromatin-bound Cdc6 and MCM proteins f rom quiescent cells appears not to be due to the absence of ORC subunits as murine homologues of Orc1 and Orc2 remain chromatin-bound in quiescent cel ls. Surprisingly, intact quiescent nuclei fail to bind exogenously added XC dc6 or to replicate in Xenopus egg extracts immunodepleted of ORC, even tho ugh G1- or S-phase nuclei still replicate in these extracts. Our results id entify Cdc6 and the MCM complex as essential replication components absent from quiescent chromatin due to nonfunctional chromatin-bound ORC proteins. These results can explain why quiescent mammalian nuclei are unable to rep licate in vivo and in Xenopus egg extracts. (C) 2000 Academic Press.