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

Citation
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
Citations number
51
Categorie Soggetti
Biochemistry & Biophysics
Journal title
JOURNAL OF BIOLOGICAL CHEMISTRY
ISSN journal
00219258 → ACNP
Volume
276
Issue
20
Year of publication
2001
Pages
17092 - 17100
Database
ISI
SICI code
0021-9258(20010518)276:20<17092:ATABTR>2.0.ZU;2-U
Abstract
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.