Mcm2, but not RPA, is a component of the mammalian early G1-phase prereplication complex.
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.
Dimitrova
D S
DS
Department of Biochemistry and Molecular Biology, S.U.N.Y. Health Science Center, Syracuse, New York 13210, USA.
Todorov
I T
IT
Melendy
T
T
Gilbert
D M
DM
D000818:Animals; D016466:CHO Cells; D002843:Chromatin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002875:Chromosomes / Q000235:genetics; D006224:Cricetinae; D004261:DNA Replication / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism*; D016193:G1 Phase / Q000235:genetics; D006219:Halogens / Q000378:metabolism; D064111:Minichromosome Maintenance Complex Component 2; D008938:Mitosis / Q000235:genetics; D009685:Nuclear Envelope / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism*; D009711:Nucleotides / Q000378:metabolism; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D011485:Protein Binding; D018741:Replication Origin / Q000235:genetics; D051716:Replication Protein A; D016196:S Phase / Q000235:genetics; D013692:Telophase / Q000235:genetics; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics; D013997:Time Factors
D002843:Chromatin; D004268:DNA-Binding Proteins; D006219:Halogens; D009687:Nuclear Proteins; D009711:Nucleotides; D018809:Proliferating Cell Nuclear Antigen; D051716:Replication Protein A; D013762:Tetrahydrofolate Dehydrogenase; D064111:Minichromosome Maintenance Complex Component 2