Isolation of the Cdc45/Mcm2–7/GINS (CMG) complex, a candidate for the eukaryotic DNA replication fork helicase

Abstract

The protein Cdc45 plays a critical but poorly understood role in the initiation and elongation stages of eukaryotic DNA replication. To study Cdc45's function in DNA replication, we purified Cdc45 protein from Drosophila embryo extracts by a combination of traditional and immunoaffinity chromatography steps and found that the protein exists in a stable, high-molecular-weight complex with the Mcm2-7 hexamer and the GINS tetramer. The purified Cdc45͞Mcm2-7͞GINS complex is associated with an active ATPdependent DNA helicase function. RNA interference knock-down experiments targeting the GINS and Cdc45 components establish that the proteins are required for the S phase transition in Drosophila cells. The data suggest that this complex forms the core helicase machinery for eukaryotic DNA replication.Drosophila ͉ ATPase

Human Mcm10 Regulates the Catalytic Subunit of DNA Polymerase-α and Prevents DNA Damage during Replication

“…A role for human Mcm10 in elongation is also consistent with previous reports that argue that Mcm10 stays associated with chromatin throughout S phase in HeLa cells (Izumi et al, 2000(Izumi et al, , 2001. Moreover, the lack of complete colocalization with BrdU-labeled DNA or PCNA has previously been documented for components of the Mcm2-7 complex (Madine et al, 1995), which constitutes the core of the replicative helicase in eukaryotic cells (Schwacha and Bell, 2001;Moyer et al, 2006). Therefore, the incomplete overlap between Mcm10 and PCNA foci does not necessarily mean that Mcm10 is not involved in elongation.…”

“…The first step is the formation of a prereplicative complex (pre-RC) (Diffley et al, 1994), which is subsequently transformed into a preinitiation complex, and finally into a pair of functional replication forks that have the ability to unwind parental DNA and synthesize new daughter strands (Mendez and Stillman, 2003). DNA unwinding is likely catalyzed by the minichromosome maintenance (Mcm) 2-7 complex (Aparicio et al, 1997;Labib et al, 2000;Shechter et al, 2004) in conjunction with two coactivators, Cdc45 and GINS (Pacek and Walter, 2004;Gambus et al, 2006;Moyer et al, 2006;Pacek et al, 2006). The association of Cdc45 and GINS with DNA is interdependent (Kubota et al, 2003;Takayama et al, 2003), and it requires yet another factor, Mcm10 (Wohlschlegel et al, 2002;Gregan et al, 2003;Sawyer et al, 2004).…”

The cell cycle and cancer

“…Cdc7 phosphorylates the Mcm2, 4 and 6 subunits, thereby inducing a conformational change that stimulates MCM helicase activity [51][52][53]. The formation of an active helicase leads to the recruitment of additional factors, including Cdc45 and the four subunit GINS complex, which is also dependent on Cdc7 kinase activity [54][55][56]. Once activated, the MCM helicase unwinds double-stranded DNA at origins to generate a single-stranded DNA template required to recruit the DNA synthesis machinery containing RPA, PCNA and DNA polymerase α-primase [46].…”

Human Cdt1 Lacking the Evolutionarily Conserved Region That Interacts with MCM2–7 Is Capable of Inducing Re-replication

“…1 and 2). MCM2-7 is thought to be the replicative helicase responsible for unwinding DNA ahead of the replication machinery (3)(4)(5)(6). It is loaded in a Cdc6-and Cdt1-dependent manner, and this loading can only occur during low levels of CDK activity, in late M phase and early G 1 phase.…”