Focus-formation of replication protein A, activation of checkpoint system and DNA repair synthesis induced by DNA double-strand breaks in Xenopus egg extract.
The response to DNA damage was analyzed using a cell-free system consisting of Xenopus egg extract and demembranated sperm nuclei. In the absence of DNA-damaging agents, detergent-resistant accumulation of replication protein A appeared in nuclei after a 30 minute incubation, and a considerable portion of the replication protein A signals disappeared during a further 30 minute incubation. Similar replication protein A accumulation was observed in the nuclei after a 30 minute incubation in the extract containing camptothecin, whereas a further 30 minute incubation generated discrete replication protein A foci. The addition of camptothecin also induced formation of gamma-H2AX foci, which have been previously shown to localize at sites of DSBs. Analysis of the time course of DNA replication and results obtained using geminin, an inhibitor of licensing for DNA replication, suggest that the discrete replication protein A foci formed in response to camptothecin-induced DNA damage occur in a DNA-replication-dependent manner. When the nuclei were incubated in the extract containing EcoRI, discrete replication protein A foci were observed at 30 minutes as well as at 60 and 90 minutes after incubation, and the focus-formation of replication protein A was not sensitive to geminin. DNA replication was almost completely inhibited in the presence of EcoRI and the inhibition was sensitive to caffeine, an inhibitor of ataxia telangiectasia mutated protein (ATM) and ATM- and Rad3-related protein (ATR). However, the focus-formation of replication protein A in the presence of EcoRI was not influenced by caffeine treatment. EcoRI-induced incorporation of biotin-dUTP into chromatin was observed following geminin-mediated inhibition of DNA replication, suggesting that the incorporation was the result of DNA repair. The biotin-dUTP signal co-localized with replication protein A foci and was not significantly suppressed or stimulated by the addition of caffeine.
Molecular Cell Biology Laboratory, Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba-ku, Sendai, Miyagi 980-8578, Japan.
Takayuki
T
Kobayashi
Shusuke
S
Tada
Takashi
T
Tsuyama
Hiromu
H
Murofushi
Masayuki
M
Seki
Takemi
T
Enomoto
D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins; D001710:Biotin / Q000031:analogs & derivatives* / Q000494:pharmacology; D002110:Caffeine / Q000494:pharmacology; D002166:Camptothecin / Q000494:pharmacology; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000494:pharmacology; D002457:Cell Extracts; D002467:Cell Nucleus / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D002474:Cell-Free System / Q000187:drug effects / Q000378:metabolism*; D004249:DNA Damage / Q000187:drug effects / Q000235:genetics*; D004260:DNA Repair / Q000187:drug effects / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000187:drug effects; D003856:Deoxyuracil Nucleotides / Q000494:pharmacology; D005057:Eukaryotic Cells / Q000187:drug effects / Q000378:metabolism*; D005260:Female; D064248:Geminin; D018816:Genes, cdc / Q000187:drug effects / Q000502:physiology*; D008297:Male; D009865:Oocytes; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D051716:Replication Protein A; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000494:pharmacology; D013094:Spermatozoa; D025521:Tumor Suppressor Proteins; D029867:Xenopus Proteins; D014982:Xenopus laevis; D014544:Uridine Triphosphate / Q000031:analogs & derivatives
D064007:Ataxia Telangiectasia Mutated Proteins; D001710:Biotin; D002110:Caffeine; D002166:Camptothecin; D018797:Cell Cycle Proteins; D002457:Cell Extracts; D004268:DNA-Binding Proteins; D003856:Deoxyuracil Nucleotides; D064248:Geminin; D017346:Protein Serine-Threonine Kinases; D051716:Replication Protein A; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); D025521:Tumor Suppressor Proteins; D029867:Xenopus Proteins; C577998:GMNN protein, Xenopus; C087624:biotin-16-dUTP; D014544:Uridine Triphosphate; C020098:DNA modification methylase EcoRI; C576898:Atr protein, Xenopus