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© The Rockefeller University Press, 0021-9525/1999//709 $5.00
The Journal of Cell Biology, Volume 146, Number 4, , 1999 709-722

Original Article

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
Department of Biochemistry and Molecular Biology, S.U.N.Y. Health Science Center, 750 East Adams Street, Syracuse, NY 13210.(315) 464-8750(315) 464-8723

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

© 1999 The Rockefeller University Press

1.used in this paper: BrdU, bromodeoxyuridine; CldU, 5-chloro-2'-deoxyuridine; DHFR, dihydrofolate reductase; FFA, focus forming activity; IdU, 5-iodo-2'-deoxyuridine; ODP, origin decision point; ORC, origin recognition complex; PCNA, proliferating cell nuclear antigen; pre-RCs, prereplication complexes or centers; RPA, replication protein A; SSB, single-stranded DNA-binding protein; TxRed, Texas Red; xlRPA, Xenopus laevis RPA homologue


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In Brief
J. Cell Biol. 1999 146: 1-2. [Full Text] [PDF]





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