Abstract
Human Rad17 (hRad17) is centrally involved in the activation of cell-cycle checkpoints by genotoxic agents or replication stress. Here we identify hMCM7, a core component of the DNA replication apparatus, as a novel hRad17-interacting protein. In HeLa cells, depletion of either hRad17 or hMCM7 with small-interfering RNA suppressed ultraviolet (UV) light- or aphidicolin-induced hChk1 phosphorylation, and abolished UV-induced S-phase checkpoint activation. Similar results were obtained after transfection of these cells with a fusion protein containing the hMCM7-binding region of hRad17. The hMCM7-depleted cells were also defective for the formation of ATR-containing nuclear foci after UV irradiation, suggesting that hMCM7 is required for stable recruitment of ATR to damaged DNA. These results demonstrate that hMCM7 plays a direct role in the transmission of DNA damage signals from active replication forks to the S-phase checkpoint machinery in human cells.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, Non-P.H.S.
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Aphidicolin / pharmacology
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Ataxia Telangiectasia Mutated Proteins
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Cell Cycle Proteins / genetics
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Cell Cycle Proteins / metabolism
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Cell Cycle Proteins / physiology*
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Cell Line, Tumor
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Checkpoint Kinase 1
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Checkpoint Kinase 2
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DNA Damage
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DNA Replication / physiology*
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DNA-Binding Proteins / genetics
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DNA-Binding Proteins / physiology*
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Green Fluorescent Proteins / genetics
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HeLa Cells
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Humans
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Minichromosome Maintenance Complex Component 7
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Nuclear Proteins / genetics
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Nuclear Proteins / physiology*
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Phosphorylation
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Protein Binding
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Protein Kinases / metabolism
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Protein Serine-Threonine Kinases / metabolism
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Recombinant Fusion Proteins / genetics
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S Phase / physiology
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Signal Transduction
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Transfection
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Two-Hybrid System Techniques
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Ultraviolet Rays
Substances
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Cell Cycle Proteins
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DNA-Binding Proteins
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Nuclear Proteins
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Rad17 protein, human
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Recombinant Fusion Proteins
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Green Fluorescent Proteins
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Aphidicolin
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Protein Kinases
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Checkpoint Kinase 2
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ATR protein, human
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Ataxia Telangiectasia Mutated Proteins
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CHEK1 protein, human
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CHEK2 protein, human
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Checkpoint Kinase 1
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Protein Serine-Threonine Kinases
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MCM7 protein, human
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Minichromosome Maintenance Complex Component 7