Abstract
We have studied the rate of DNA synthesis, cell cycle distribution, formation of gamma-H2AX, and Rad51 nuclear foci and association of Rad51 with the nuclear matrix after treatment of HeLa cells with the interstrand crosslinking agent mitomycin C (MMC) in the presence of the kinase inhibitors caffeine and wortmannin. The results showed that MMC treatment arrested the cells in S-phase and induced the appearance of gamma-H2AX and Rad51 nuclear foci, accompanied with a sequestering of Rad51 to the nuclear matrix. These effects were abrogated by caffeine, which inhibits the Ataxia-telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR) kinases. However, wortmannin at a concentration that inhibits ATM, but not ATR did not affect cell cycle progression, damage-induced phosphorylation of H2AX and Rad51 foci formation, and association with the nuclear matrix, suggesting that the S-phase arrest induced by MMC is ATR-dependent. These findings were confirmed by experiments with ATR-deficient and AT cells. They indicate that the DNA damage ATR-dependent S-phase checkpoint pathway may regulate the spatiotemporal organization of the process of repair of interstrand crosslinks.
(c) 2007 Wiley-Liss, Inc.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Androstadienes / pharmacology
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Antibiotics, Antineoplastic / pharmacology*
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Ataxia Telangiectasia / genetics
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Ataxia Telangiectasia / metabolism
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Ataxia Telangiectasia / pathology
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Ataxia Telangiectasia Mutated Proteins
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Caffeine / pharmacology
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Cell Cycle Proteins / antagonists & inhibitors
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Cell Cycle Proteins / metabolism*
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Cell Nucleus / drug effects*
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Cell Nucleus / metabolism
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Chromatin / metabolism
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Cross-Linking Reagents / pharmacology*
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DNA Damage / drug effects*
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DNA Replication / drug effects
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DNA-Binding Proteins / antagonists & inhibitors
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DNA-Binding Proteins / metabolism*
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HeLa Cells
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Histones / metabolism
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Humans
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Mitomycin / pharmacology*
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Nuclear Matrix / metabolism
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Phosphorylation
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Protein Kinase Inhibitors / pharmacology
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Protein Serine-Threonine Kinases / antagonists & inhibitors
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Protein Serine-Threonine Kinases / deficiency
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Protein Serine-Threonine Kinases / metabolism*
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Rad51 Recombinase / metabolism
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Recombination, Genetic / drug effects
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S Phase / drug effects*
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Time Factors
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Tumor Suppressor Proteins / antagonists & inhibitors
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Tumor Suppressor Proteins / metabolism*
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Wortmannin
Substances
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Androstadienes
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Antibiotics, Antineoplastic
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Cell Cycle Proteins
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Chromatin
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Cross-Linking Reagents
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DNA-Binding Proteins
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H2AX protein, human
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Histones
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Protein Kinase Inhibitors
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Tumor Suppressor Proteins
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Caffeine
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Mitomycin
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ATM protein, human
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ATR protein, human
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Ataxia Telangiectasia Mutated Proteins
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Protein Serine-Threonine Kinases
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RAD51 protein, human
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Rad51 Recombinase
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Wortmannin