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J. Cell Biol., Volume 146, Number 4, August 23, 1999 709-722
Copyright © 1999 by The Rockefeller University Press.

Mcm2, but Not RPA, Is a Component of the Mammalian Early G1-Phase Prereplication Complex

Daniela S. Dimitrovaa, Ivan T. Todorovb, Thomas Melendyc, and David M. Gilberta
a Department of Biochemistry and Molecular Biology, S.U.N.Y. Health Science Center, Syracuse, New York 13210
b Desmos, Inc., San Diego, California 92121
c Department of Microbiology, S.U.N.Y. at Buffalo, School of Medicine and Biomedical Sciences, Buffalo, New York 14214

Correspondence to: David M. Gilbert, Department of Biochemistry and Molecular Biology, S.U.N.Y. Health Science Center, 750 East Adams Street, Syracuse, NY 13210. Tel:(315) 464-8723 Fax:(315) 464-8750 E-mail:gilbertd{at}vax.cs.hscsyr.edu.

Previous experiments in Xenopus egg extracts identified what appeared to be two independently assembled prereplication complexes (pre-RCs) for DNA replication: the stepwise assembly of ORC, Cdc6, and Mcm onto chromatin, and the FFA-1–mediated recruitment of RPA into foci on chromatin. We have investigated whether both of these pre-RCs can be detected in Chinese hamster ovary (CHO) cells. Early- and late-replicating chromosomal domains were pulse-labeled with halogenated nucleotides and prelabeled cells were synchronized at various times during the following G1-phase. The recruitment of Mcm2 and RPA to these domains was examined in relation to the formation of a nuclear envelope, specification of the dihydrofolate reductase (DHFR) replication origin and entry into S-phase. Mcm2 was loaded gradually and cumulatively onto both early- and late-replicating chromatin from late telophase throughout G1-phase. During S-phase, detectable Mcm2 was rapidly excluded from PCNA-containing active replication forks. By contrast, detergent-resistant RPA foci were undetectable until the onset of S-phase, when RPA joined only the earliest-firing replicons. During S-phase, RPA was present with PCNA specifically at active replication forks. Together, our data are consistent with a role for Mcm proteins, but not RPA, in the formation of mammalian pre-RCs during early G1-phase.

Key Words: pre-RC, Mcm, RPA, cell cycle, DNA replication domains


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