Activation of the S phase DNA damage checkpoint by mitomycin C.
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.
Mladenov
Emil
E
Institute of Molecular Biology, Bulgarian Academy of Sciences, Sofia, Bulgaria.
Tsaneva
Irina
I
Anachkova
Boyka
B
D000730:Androstadienes / Q000494:pharmacology; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D001260:Ataxia Telangiectasia / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D064007:Ataxia Telangiectasia Mutated Proteins; D002110:Caffeine / Q000494:pharmacology; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002467:Cell Nucleus / Q000187:drug effects* / Q000378:metabolism; D002843:Chromatin / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D004249:DNA Damage / Q000187:drug effects*; D004261:DNA Replication / Q000187:drug effects; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006367:HeLa Cells; D006657:Histones / Q000378:metabolism; D006801:Humans; D016685:Mitomycin / Q000494:pharmacology*; D015530:Nuclear Matrix / Q000378:metabolism; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000172:deficiency / Q000378:metabolism*; D051135:Rad51 Recombinase / Q000378:metabolism; D011995:Recombination, Genetic / Q000187:drug effects; D016196:S Phase / Q000187:drug effects*; D013997:Time Factors; D025521:Tumor Suppressor Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000077191:Wortmannin
D000730:Androstadienes; D000903:Antibiotics, Antineoplastic; D018797:Cell Cycle Proteins; D002843:Chromatin; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; C468783:H2AX protein, human; D006657:Histones; D047428:Protein Kinase Inhibitors; D025521:Tumor Suppressor Proteins; D002110:Caffeine; D016685:Mitomycin; C576324:ATM protein, human; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases; C495153:RAD51 protein, human; D051135:Rad51 Recombinase; D000077191:Wortmannin