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A more recent version of this article appeared on May 11, 2001
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Papers In Press, published online ahead of print March 6, 2001
J. Biol. Chem, 10.1074/jbc.M100271200
Submitted on January 11, 2001
Revised on March 5, 2001
Accepted on March 5, 2001

Aphidicolin triggers a block to replication origin firing in Xenopus egg extracts

Kathrin Marheineke and Olivier Hyrien

Laboratoire de Génétique Moléculaire, Ecole Normale Supérieure, 75230, Paris Cedex 05

Corresponding Author: hyrien{at}wotan.ens.fr

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 S phase in Xenopus egg extracts. In order to study the temporal regulation of origin firing, we have analysed 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, 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 through S phase. Therefore, initiation during 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.


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