Abstract
We have cloned a Xenopus Dbf4-related factor named Drf1 and characterized this protein by using Xenopus egg extracts. Drf1 forms an active complex with the kinase Cdc7. However, most of the Cdc7 in egg extracts is not associated with Drf1, which raises the possibility that some or all of the remaining Cdc7 is bound to another Dbf4-related protein. Immunodepletion of Drf1 does not prevent DNA replication in egg extracts. Consistent with this observation, Cdc45 can still associate with chromatin in Drf1-depleted extracts, albeit at significantly reduced levels. Nonetheless, Drf1 displays highly regulated binding to replicating chromatin. Treatment of egg extracts with aphidicolin results in a substantial accumulation of Drf1 on chromatin. This accumulation is blocked by addition of caffeine and by immunodepletion of either ATR or Claspin. These observations suggest that the increased binding of Drf1 to aphidicolin-treated chromatin is an active process that is mediated by a caffeine-sensitive checkpoint pathway containing ATR and Claspin. Abrogation of this pathway also leads to a large increase in the binding of Cdc45 to chromatin. This increase is substantially reduced in the absence of Drf1, which suggests that regulation of Drf1 might be involved in the suppression of Cdc45 loading during replication arrest. We also provide evidence that elimination of this checkpoint causes resumed initiation of DNA replication in both Xenopus tissue culture cells and egg extracts. Taken together, these observations argue that Drf1 is regulated by an intra-S-phase checkpoint mechanism that down-regulates the loading of Cdc45 onto chromatin containing DNA replication blocks.
Highlights
In eukaryotes, DNA replication is initiated by a multistep process
Treatment of egg extracts with aphidicolin results in a substantial accumulation of Dbf4-related factor 1 (Drf1) on chromatin. This accumulation is blocked by addition of caffeine and by immunodepletion of either ATR or Claspin. These observations suggest that the increased binding of Drf1 to aphidicolin-treated chromatin is an active process that is mediated by a caffeine-sensitive checkpoint pathway containing ATR and Claspin
The cDNA that we have identified in Xenopus encodes a 772amino acid protein that is homologous to both human proteins (32 and 26% identical to human Drf1 and ASK, respectively) but shares a higher identity with Drf1 (Fig. 1A)
Summary
DNA replication is initiated by a multistep process. Early in the G1-phase, replication initiation factors are sequentially assembled onto replication origins to form prereplicative complexes (pre-RCs).1 At the core of the pre-RC is. These observations suggest that the increased binding of Drf1 to aphidicolin-treated chromatin is an active process that is mediated by a caffeine-sensitive checkpoint pathway containing ATR and Claspin. Chromatin Binding of Drf1 and Cdc7 following Aphidicolin Treatment Is Checkpoint-regulated—Because Cdc7 and Dbf4 are regulated by the DNA replication checkpoint in Xenopus and in yeast, we tested whether Drf1 may have a checkpoint-specific function.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
