Aphidicolin Triggers a Block to Replication Origin Firing inXenopus Egg Extracts

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 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.

Xenopus CENP-A assembly into chromatin requires base excision repair proteins

“…Aphidicolin is an inhibitor of DNA polymerase alpha that blocks both the elongation and initiation phases of DNA replication in Xenopus egg extracts [18]. We observed that addition of aphidicolin to extracts caused a three-fold increase in the number of CENP-A foci at 60 or 90 minutes, relative to untreated sperm nuclei ( Figure 3A, B and H).…”

Mcm2 Is a Direct Substrate of ATM and ATR during DNA Damage and DNA Replication Checkpoint Responses

“…The presence of double-stranded DNA breaks in egg extracts inhibits chromosomal DNA replication as well as entry into mitosis (17)(18)(19). Interestingly, aphidicolin, which inhibits DNA polymerase activity, also triggers a pathway that blocks utilization of as yet unfired origins (24,44). This response is sensitive to the checkpoint-overriding compound caffeine.…”

Defining replication origin efficiency using DNA fiber assays

“…In this approach, an asynchronous population of cells is pulsed-labeled for a short period of time with halogenated nucleotides to determine the position of replication origins (arrowheads). DNA fibers can be counterstained with anti-ssDNA antibodies (blue) (Versini et al 2003), and specific regions can be analyzed by FISH (Lebofsky et al 2006); c DNA combing analysis of densely spaced origins in Xenopus egg extracts (Marheineke and Hyrien 2001). Initiation sites are labeled with DIG-dUTP (red) to mark replication origins (arrowheads) after release from an aphidicolin block and biotin-dUTP (green) is added at variable times until completion of DNA replication; d DNA combing analysis of interorigin distances (IODs) in yeast.…”