Abstract
Mcm4/6/7 forms a complex possessing DNA helicase activity, suggesting that Mcm may be a central component for the replicative helicase. Although Cdt1 is known to be essential for loading of Mcm onto the chromatin, its precise role in pre-RC formation and replication initiation is unknown. Using purified proteins, we show that Cdt1 forms a complex with Mcm4/6/7, Mcm2/3/4/5/6/7, and Mcm2/4/6/7 in glycerol gradient fractionation through interaction with Mcm2 and Mcm4/6. In the glycerol gradient fractionation, Mcm4/6/7-Cdt1 forms a complex (speculated to be a (Mcm4/6/7)2-Cdt13 assembly) in the presence of ATP, which is significantly larger than the Mcm4/6/7-Cdt1 complex generated in its absence. Furthermore, DNA binding and helicase activities of Mcm4/6/7 are significantly stimulated by Cdt1 protein in vitro. We generated a Cdt1 mutant, which fails to stimulate DNA binding and helicase activities of Mcm4/6/7. This mutant Cdt1 showed reduced interaction with Mcm and is deficient in the formation of a high molecular weight complex with Mcm. Thus, a productive interaction between Cdt1 and MCM appears to be essential for efficient loading of MCM onto template DNA, as well as for the efficient unwinding reaction.
MeSH terms
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Adenosine Triphosphate / chemistry
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Adenosine Triphosphate / metabolism
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Animals
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Cell Cycle Proteins / chemistry
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Cell Cycle Proteins / genetics
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Cell Cycle Proteins / metabolism*
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Cell-Free System / metabolism
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DNA / chemistry
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DNA / genetics
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DNA / metabolism*
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DNA Helicases / chemistry
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DNA Helicases / genetics
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DNA Helicases / metabolism*
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DNA-Binding Proteins / chemistry
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DNA-Binding Proteins / genetics
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DNA-Binding Proteins / metabolism*
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Mice
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Minichromosome Maintenance Complex Component 4
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Minichromosome Maintenance Complex Component 6
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Minichromosome Maintenance Complex Component 7
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Multiprotein Complexes / chemistry
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Multiprotein Complexes / genetics
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Multiprotein Complexes / metabolism*
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Mutation
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Nuclear Proteins / chemistry
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Nuclear Proteins / genetics
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Nuclear Proteins / metabolism*
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Protein Binding / physiology
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Recombinant Proteins / chemistry
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Recombinant Proteins / genetics
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Recombinant Proteins / metabolism
Substances
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Cell Cycle Proteins
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DNA-Binding Proteins
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Multiprotein Complexes
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Nuclear Proteins
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Recombinant Proteins
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Ris2 protein, mouse
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Adenosine Triphosphate
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DNA
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DNA Helicases
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Mcm4 protein, mouse
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Mcm6 protein, mouse
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Mcm7 protein, mouse
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Minichromosome Maintenance Complex Component 4
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Minichromosome Maintenance Complex Component 6
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Minichromosome Maintenance Complex Component 7