Biochemical Analysis of the Intrinsic Mcm4-Mcm6-Mcm7 DNA Helicase Activity
TLDR
Biochemical analyses of various mutant Mcm4,6,7 complexes indicated that the ATP binding activity of the Mcm6 protein in the complex is critical for DNA helicase activity and that theMcm4 protein may play a role in the single-stranded DNA bindingActivity of the complex.Abstract:
Mcm proteins play an essential role in eukaryotic DNA replication, but their biochemical functions are poorly understood. Recently, we reported that a DNA helicase activity is associated with an Mcm4-Mcm6-Mcm7 (Mcm4,6,7) complex, suggesting that this complex is involved in the initiation of DNA replication as a DNA-unwinding enzyme. In this study, we have expressed and isolated the mouse Mcm2,4,6,7 proteins from insect cells and characterized various mutant Mcm4,6,7 complexes in which the conserved ATPase motifs of the Mcm4 and Mcm6 proteins were mutated. The activities associated with such preparations demonstrated that the DNA helicase activity is intrinsically associated with the Mcm4,6,7 complex. Biochemical analyses of these mutant Mcm4,6,7 complexes indicated that the ATP binding activity of the Mcm6 protein in the complex is critical for DNA helicase activity and that the Mcm4 protein may play a role in the single-stranded DNA binding activity of the complex. The results also indicated that the two activities of DNA helicase and single-stranded DNA binding can be separated.read more
Citations
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DNA replication in eukaryotic cells
Stephen P. Bell,Anindya Dutta +1 more
TL;DR: This review describes the current understanding of the events of initiation of eukaryotic replication factors and how they are coordinated with cell cycle progression and emphasizes recent progress in determining the function of the different replication factors once they have been assembled at the origin.
Journal ArticleDOI
AAA+ superfamily ATPases: common structure--diverse function.
Teru Ogura,Anthony J. Wilkinson +1 more
TL;DR: The AAA+ proteins represent a novel type of molecular chaperone and are versatile in their mode of action, which collectively seem to involve some form of disruption of molecular or macromolecular structure.
Journal ArticleDOI
Isolation of the Cdc45/Mcm2–7/GINS (CMG) complex, a candidate for the eukaryotic DNA replication fork helicase
TL;DR: 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, and suggest that this complex forms the core helicase machinery for eukaryotic DNA replication.
Journal ArticleDOI
Uninterrupted MCM2-7 Function Required for DNA Replication Fork Progression
TL;DR: It is shown that depletion of MCMs after initiation irreversibly blocks the progression of replication forks in Saccharomyces cerevisiae and restricts MCM loading to the G(1) phase ensures that initiation and elongation occur just once per cell cycle.
Journal ArticleDOI
Eukaryotic MCM Proteins: Beyond Replication Initiation
TL;DR: Analysis of mcm mutant phenotypes and interactions with other factors have implicated the MCM proteins in other chromosome transactions including damage response, transcription, and chromatin structure, indicating that the MCMs are central players in many aspects of genome stability.
References
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Journal ArticleDOI
Components and Dynamics of DNA Replication Complexes in S. cerevisiae: Redistribution of MCM Proteins and Cdc45p during S Phase
TL;DR: The results identify protein components of the pre-RC and a novel replication complex appearing at the G1/S transition (the RC), and suggest that after initiation MCM proteins and Cdc45p move with eukaryotic replication forks.
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Mechanisms of helicase-catalyzed DNA unwinding.
TL;DR: This review discusses mechanistic aspects of helicase-catalyzed DNA unwinding and translocation with a focus on energetic (thermodynamic), kinetic, and structural studies of the few DNA helicases for which such information is available.
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Cdc6p-dependent loading of Mcm proteins onto pre-replicative chromatin in budding yeast
TL;DR: The Mcm proteins of budding yeast are abundant and are quantitatively found in a chromatin-enriched fraction specifically during the G1 phase of the cell cycle, providing evidence that a conserved biochemical pathway plays a critical role in coordinating DNA replication with mitosis in both yeast and higher eukaryotes.
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Loading of an Mcm Protein onto DNA Replication Origins Is Regulated by Cdc6p and CDKs
TL;DR: The data suggest that the loading of Mcm proteins onto origins is regulated by two mechanisms: first, by Cdc6p occupancy, and second, by S- and M-CDKs, whose activity during S, G2, and M phases prevents Mcm loading.
Journal ArticleDOI
A DNA Helicase Activity Is Associated with an MCM4, -6, and -7 Protein Complex *
TL;DR: Results suggest that this helicase may be involved in the initiation of DNA replication as a DNA unwinding enzyme.