K. Marheineke et O. Hyrien, Aphidicolin triggers a block to replication origin firing in Xenopus egg extracts, J BIOL CHEM, 276(20), 2001, pp. 17092-17100
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 analy
zed origin activation in sperm nuclei treated with the DNA polymerase inhib
itor aphidicolin, Sperm chromatin was incubated in Xenopus egg extracts in
the presence of aphidicolin and transferred to a fresh extract, and digoxig
enin-dUTP and biotin-dUTP were added at various times after aphidicolin rel
ease to selectively label early and late replicating DNA. Molecular combing
analysis of single DNA fibers showed that only a fraction of potential ori
gins were able to initiate in the presence of aphidicolin, After release fr
om aphidicolin, the remaining origins fired asynchronously throughout the S
phase. Therefore, initiation during the S phase depends on the normal prog
ression of replication forks assembled at earlier activated origins. Caffei
ne, an inhibitor of the checkpoint kinases ATR and ATM, did not relieve the
aphidicolin-induced block to origin firing. We conclude that a caffeine-in
sensitive intra-S phase checkpoint regulates origin activation when DNA syn
thesis is inhibited in Xenopus egg extracts.