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Mcm2, but not RPA, is a component of the mammalian early G1-phase prereplication complex
D S Dimitrova1, I T Todorov, T Melendy
1Department of Biochemistry and Molecular Biology, S.U.N.Y. Health Science Center, Syracuse, New York 13210, USA.
Abstract:
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.
Insights
In Chinese hamster ovary cells, Mcm proteins, not RPA, are crucial for forming prereplication complexes (pre-RCs) during early G1 phase, aiding DNA replication initiation.
Area of Science:
- Cell Biology
- Molecular Biology
- Genetics
Background:
- Prereplication complexes (pre-RCs) are essential for DNA replication.
- Previous Xenopus studies suggested two independent pre-RC assembly pathways involving Mcm and RPA proteins.
Purpose of the Study:
- To investigate the presence and roles of Mcm and RPA in pre-RC formation in mammalian cells (CHO cells).
- To determine the timing and localization of Mcm and RPA recruitment during the cell cycle.
Main Methods:
- Chinese hamster ovary (CHO) cells were used.
- Chromosomal domains were pulse-labeled with halogenated nucleotides.
- Cells were synchronized at different G1-phase stages.
- Recruitment of Mcm2 and RPA was examined using techniques like PCNA staining and nuclear envelope formation analysis.
Main Results:
- Mcm2 gradually loaded onto chromatin from late telophase through G1 phase.
- RPA foci appeared only at the onset of S-phase, localizing to active replication forks.
- Mcm2 was excluded from active replication forks during S-phase, while RPA was present.
Conclusions:
- Mammalian pre-RC formation involves Mcm proteins during early G1 phase.
- RPA is not involved in the initial formation of mammalian pre-RCs but functions during S-phase at active replication sites.