Cdc45-MCM-GINS, a new power player for DNA replication

Aparicio, Tomás; Ibarra, Arkaitz; Méndez, Juan Pablo

doi:10.1186/1747-1028-1-18

Cited by 62 publications

(36 citation statements)

References 36 publications

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“…The results were plotted as fold competition, where a 10-fold competition corresponds to a 90% reduction in MCM binding to the labeled oligonucleotide. B, the addition of 100-fold molar excess of unlabeled poly(dT) competitor ssDNA oligonucleotides of length 15,20,25,30,35,40,50,60, or 80 nt to standard binding reactions containing radiolabeled poly(dT) 80mer. C, the indicated amount of unlabeled dsDNA substrate was added to standard ssDNA binding reactions (4 nM 32 P-labeled oligonucleotide 510, 120 nM MCM hexamer, 5 mM ATP␥S, final volume of 12.5 l).…”

Section: Mcm2-7 and Mcm467 Associate Differently With Ssdna; Mcm2-7 Hmentioning

confidence: 99%

“…The discrepancy between the in vivo involvement of all six subunits in DNA replication and the in vitro participation of only a specific subgroup of MCM subunits in DNA unwinding remains unexplained. Recently, MCM2-7 has been isolated in vivo as part of a larger macromolecular complex having ATPdependent helicase activity; this complex additionally contains the essential GINS complex and CDC45, suggesting that these factors are activators of MCM helicase activity (20).Although MCM467 has been extensively characterized biochemically, little work has been done with the MCM2-7 heterohexamer. To determine why the MCM2-7 complex lacks * This work was supported by Grant RSG-05-113-01-CCG from the American Cancer Society (to A. S.).…”

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Differences in the Single-stranded DNA Binding Activities of MCM2-7 and MCM467

Bochman

Schwacha

2007

Journal of Biological Chemistry

104

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The MCM2-7 complex, a hexamer containing six distinct and essential subunits, is postulated to be the eukaryotic replicative DNA helicase. Although all six subunits function at the replication fork, only a specific subcomplex consisting of the MCM4, 6, and 7 subunits (MCM467) and not the MCM2-7 complex exhibits DNA helicase activity in vitro. To understand why MCM2-7 lacks helicase activity and to address the possible function of the MCM2, 3, and 5 subunits, we have compared the biochemical properties of the Saccharomyces cerevisiae MCM2-7 and MCM467 complexes. We demonstrate that both complexes are toroidal and possess a similar ATP-dependent single-stranded DNA (ssDNA) binding activity, indicating that the lack of helicase activity by MCM2-7 is not due to ineffective ssDNA binding. We identify two important differences between them. MCM467 binds dsDNA better than MCM2-7. In addition, we find that the rate of MCM2-7/ssDNA association is slow compared with MCM467; the association rate can be dramatically increased either by preincubation with ATP or by inclusion of mutations that ablate the MCM2/5 active site. We propose that the DNA binding differences between MCM2-7 and MCM467 correspond to a conformational change at the MCM2/5 active site with putative regulatory significance.Cellular DNA is double-stranded, yet during DNA replication it must be separated into component single strands. Although DNA polymerases require a single-stranded DNA (ssDNA) 2 template for activity, they have little or no intrinsic ability to unwind double-stranded DNA (dsDNA; reviewed in Ref. 1). DNA unwinding requires an ATP-dependent molecular motor termed the replicative helicase (reviewed in Ref. 2). In both prokaryotes and eukaryotes, the loading and activation of this helicase is a central and limiting event during DNA replication. Initiation culminates in replicative helicase loading, whereas the start of elongation requires extensive separation of duplex DNA by the helicase (reviewed in Refs. 3 and 4). Despite the critical importance of the replicative helicase, both its exact identity and mechanism remain controversial in eukaryotes.Numerous studies implicate the minichromosome maintenance proteins (MCMs) as the replicative helicase. The MCMs are evolutionarily conserved from archaea to eukaryotes, with the archaea usually having a single MCM gene (5) and eukaryotes having six distinct and essential MCM genes (reviewed in Ref. 6). Each MCM protein (numbered 2-7) is an AAA ϩ ATPase, whose members include DNA helicases such as SV40 large T antigen and the papilloma virus E1 protein (7). Similar to prokaryotic replicative helicases (reviewed in Ref. 8), the six MCM subunits are both physically present in initiation and elongation complexes and functionally essential for both phases of DNA replication, evidence strongly suggesting that all six MCM subunits unwind DNA at the replication fork (reviewed in Ref.3).Despite in vivo similarities to other replicative helicases, biochemical examination of the MCM complex has provided confo...

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Section: Mcm2-7 and Mcm467 Associate Differently With Ssdna; Mcm2-7 Hmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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Differences in the Single-stranded DNA Binding Activities of MCM2-7 and MCM467

Bochman

Schwacha

2007

Journal of Biological Chemistry

104

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“…As cells enter S phase, activation of two S-phase-specific protein kinases-Clb5,6/Cdc28 (S-CDK) and the Dbf4-dependent kinase (DDK), Cdc7-triggers initiation of replication at pre-RCs. S-CDK and DDK stimulate a cascade of initiation factor interactions that results in the activation of the Mcm2-7 helicase (for review, see Aparicio et al 2006;Labib and Gambus 2007). Once activated, Mcm2-7 unwinds origin DNA to provide the ssDNA template required to recruit the remaining DNA synthesis machinery and to assemble a pair of bidirectional replication forks (Takeda and Dutta 2005).…”

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confidence: 99%

Incorporation into the prereplicative complex activates the Mcm2–7 helicase for Cdc7–Dbf4 phosphorylation

Francis¹,

Randell²,

Takara³

et al. 2009

Genes Dev.

126

123

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The essential S-phase kinase Cdc7-Dbf4 acts at eukaryotic origins of replication to trigger a cascade of protein associations that activate the Mcm2-7 replicative helicase. Also known as Dbf4-dependent kinase (DDK), this kinase preferentially targets chromatin-associated Mcm2-7 complexes that are assembled on the DNA during prereplicative complex (pre-RC) formation. Here we address the mechanisms that control the specificity of DDK action. We show that incorporation of Mcm2-7 into the pre-RC increased the level and changes the specificity of DDK phosphorylation of this complex. In the context of the pre-RC, DDK preferentially targets a conformationally distinct subpopulation of Mcm2-7 complexes that is tightly linked to the origin DNA. This targeting requires DDK to tightly associate with Mcm2-7 complexes in a Dbf4-dependent manner. Importantly, we find that DDK association with and phosphorylation of origin-linked Mcm2-7 complexes require prior phosphorylation of the pre-RC. Our findings provide insights into the mechanisms that ensure that DDK action is spatially and temporally restricted to the origin-bound Mcm2-7 complexes that will drive replication fork movement during S phase and suggest new mechanisms to regulate origin activity.[Keywords: Cdc7; DNA replication; Dbf4; Mcm2-7; kinase targeting; prereplication complex] Supplemental material is available at http://www.genesdev.org.

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“…During S phase, Mcm2-7 is activated and recruits Cdc45, the GINS complex and other Replisome Progression Complex proteins including Ctf4, Mrc1, Tof1, and Csm3 [2]. The Cdc45-MCM-GINS complex forms an active replicative helicase which unwinds the DNA, enabling its replication by DNA polymerases a, d, and e and accessory factors such as PCNA, RPA, and replication factor C (RFC) [1,3,4]. At least 70 different proteins are involved in the DNA replication process alone, and of course many other components contribute to the various cellular pathways that ensure chromosome stability.…”

Section: Dna Replication Proteinsmentioning

confidence: 99%