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 rep lication: the stepwise assembly of ORC, Cdc6, and Mcm onto chromatin, and t he FFA-l-mediated recruitment of RPA into foci on chromatin, We have invest igated whether both of these pre-RCs can be detected in Chinese hamster ova ry (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 an d RPA to these domains was examined in relation to the formation of a nucle ar envelope, specification of the dihydrofolate reductase (DHFR) replicatio n origin and entry into S-phase, Mcm2 was loaded gradually and cumulatively onto both early- and late-replicating chromatin from late telophase throug hout G1-phase, During S-phase, detectable Mcm2 was rapidly excluded from PC NA-containing active replication forks, By contrast, detergent-resistant RP A foci were undetectable until the onset of S-phase, when RPA joined only t he earliest-firing replicons, During S-phase, RPA was present with PCNA spe cifically at active replication forks, Together, our data are consistent wi th a role for Mcm proteins, but not RPA, in the formation of mammalian pre- RCs during early G1-phase.