Abstract
In eukaryotes, although the Mcm2-7 complex is a key component of the replicative DNA helicase, its association with Cdc45 and GINS (the CMG complex) is required for the activation of the DNA helicase. Here, we show that the CMG complex is localized to chromatin in human cells and describe the biochemical properties of the human CMG complex purified from baculovirus-infected Sf9 cells. The isolated complex binds to ssDNA regions in the presence of magnesium and ATP (or a nonhydrolyzable ATP analog), contains maximal DNA helicase in the presence of forked DNA structures, and translocates along the leading strand (3' to 5' direction). The complex hydrolyses ATP in the absence of DNA; unwinds duplex regions up to 500 bp; and either replication protein A or Escherichia coli single stranded binding protein increases the efficiency of displacement of long duplex regions. Using a 200-nt primed circular DNA substrate, the combined action of human DNA polymerase ε and the human CMG complex leads to the formation of products >10 kb in length. These findings suggest that the coordinated action of these replication complexes supports leading strand synthesis.
Publication types
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Research Support, N.I.H., Extramural
MeSH terms
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Adenosine Triphosphate / metabolism
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Animals
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Blotting, Western
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Cell Cycle Proteins / genetics
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Cell Cycle Proteins / metabolism*
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Cell Line
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Chromatin / genetics
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Chromatin / metabolism
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Chromosomal Proteins, Non-Histone / genetics
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Chromosomal Proteins, Non-Histone / 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 / genetics
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DNA Helicases / metabolism
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DNA Polymerase II / metabolism*
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DNA, Single-Stranded / genetics
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DNA, Single-Stranded / metabolism
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Electrophoretic Mobility Shift Assay
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HEK293 Cells
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HeLa Cells
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Humans
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Kinetics
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Minichromosome Maintenance Complex Component 2
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Nuclear Proteins / genetics
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Nuclear Proteins / metabolism*
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Nucleic Acid Conformation
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Oligonucleotides / genetics
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Oligonucleotides / metabolism
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Origin Recognition Complex / genetics
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Origin Recognition Complex / metabolism
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Protein Binding
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Spodoptera
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Substrate Specificity
Substances
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CDC45 protein, human
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Cell Cycle Proteins
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Chromatin
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Chromosomal Proteins, Non-Histone
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DNA, Single-Stranded
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GINS2 protein, human
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Nuclear Proteins
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Oligonucleotides
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Origin Recognition Complex
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Adenosine Triphosphate
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DNA
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DNA Polymerase II
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DNA Helicases
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MCM2 protein, human
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Minichromosome Maintenance Complex Component 2