Aphidicolin triggers a block to replication origin firing in Xenopus egg extracts.
DNA replication origins are located at random with respect to DNA sequence in Xenopus early embryos and on DNA replicated in Xenopus egg extracts. We have recently shown that origins fire throughout the S phase in Xenopus egg extracts. To study the temporal regulation of origin firing, we have analyzed origin activation in sperm nuclei treated with the DNA polymerase inhibitor aphidicolin. Sperm chromatin was incubated in Xenopus egg extracts in the presence of aphidicolin and transferred to a fresh extract, and digoxigenin-dUTP and biotin-dUTP were added at various times after aphidicolin release to selectively label early and late replicating DNA. Molecular combing analysis of single DNA fibers showed that only a fraction of potential origins were able to initiate in the presence of aphidicolin. After release from aphidicolin, the remaining origins fired asynchronously throughout the S phase. Therefore, initiation during the S phase depends on the normal progression of replication forks assembled at earlier activated origins. Caffeine, an inhibitor of the checkpoint kinases ATR and ATM, did not relieve the aphidicolin-induced block to origin firing. We conclude that a caffeine-insensitive intra-S phase checkpoint regulates origin activation when DNA synthesis is inhibited in Xenopus egg extracts.
Laboratoire de Génétique Moléculaire, Ecole Normale Supérieure, 46 rue d'Ulm, 75230 Paris Cedex 05, France.
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Marheineke
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Hyrien
D000818:Animals; D016590:Aphidicolin / Q000494:pharmacology*; D002110:Caffeine / Q000494:pharmacology; D002467:Cell Nucleus / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D002843:Chromatin / Q000502:physiology*; D004261:DNA Replication / Q000187:drug effects / Q000502:physiology*; D005260:Female; D007700:Kinetics; D008297:Male; D019384:Nucleic Acid Synthesis Inhibitors; D009865:Oocytes / Q000502:physiology*; D018741:Replication Origin / Q000187:drug effects / Q000502:physiology*; D016196:S Phase; D013094:Spermatozoa / Q000502:physiology*; D014020:Tissue Extracts / Q000378:metabolism; D014982:Xenopus laevis
D002843:Chromatin; D019384:Nucleic Acid Synthesis Inhibitors; D014020:Tissue Extracts; D016590:Aphidicolin; D002110:Caffeine