Xenopus Drf1, a regulator of Cdc7, displays checkpoint-dependent accumulation on chromatin during an S-phase arrest.
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.
Yanow
Stephanie K
SK
Division of Biology, Howard Hughes Medical Institute, California Institute of Technology, Pasadena, California 91125, USA.
Gold
Daniel A
DA
Yoo
Hae Yong
HY
Dunphy
William G
WG
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D000963:Antimetabolites / Q000494:pharmacology; D016590:Aphidicolin / Q000494:pharmacology; D064007:Ataxia Telangiectasia Mutated Proteins; D001973:Bromodeoxyuridine / Q000494:pharmacology; D002110:Caffeine / Q000494:pharmacology; D002352:Carrier Proteins / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000378:metabolism* / Q000502:physiology*; D000697:Central Nervous System Stimulants / Q000494:pharmacology; D002843:Chromatin / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins; D015536:Down-Regulation; D000080865:Formins; D005982:Glutathione Transferase / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism / Q000502:physiology*; D010802:Phylogeny; D011233:Precipitin Tests; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016196:S Phase; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014981:Xenopus; D029867:Xenopus Proteins
D048868:Adaptor Proteins, Signal Transducing; D000963:Antimetabolites; C102209:CDC45 protein, S cerevisiae; C416368:CLSPN protein, Xenopus; C488158:CLSPN protein, human; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D000697:Central Nervous System Stimulants; D002843:Chromatin; C108962:DIAPH1 protein, human; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D000080865:Formins; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D029867:Xenopus Proteins; D016590:Aphidicolin; D002110:Caffeine; D004247:DNA; D005982:Glutathione Transferase; C576898:Atr protein, Xenopus; C489404:CDC7 protein, S cerevisiae; C489405:CDC7 protein, Xenopus; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases; D001973:Bromodeoxyuridine