Xenopus Drf1, a Regulator of Cdc7, Displays Checkpoint-dependent Accumulation on Chromatin during an S-phase Arrest

Creators: Yanow, Stephanie K.; Gold, Daniel A.; Yoo, Hae Yong; Dunphy, William G.

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.

Additional Information

© 2003 the American Society for Biochemistry and Molecular Biology. Received for publication, July 3, 2003 , and in revised form, July 31, 2003. Originally published In Press as doi:10.1074/jbc.M307144200 on August 1, 2003. We are grateful to J. Blow, A. Kumagai, and J. Lee for generously providing anti-Mcm4, anti-ATR, and anti-Cdc45 antibodies, respectively. We also thank J. Blow for valuable suggestions regarding alkaline agarose gels. We thank all members of the laboratory for criticisms and comments during the preparation of the manuscript. The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY328889. This work was supported in part by a grant from the National Institutes of Health (to W. G. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. [S.K.Y. was] [s]upported by a postdoctoral fellowship from the California Institute of Technology. {D.A.G. was] [s]upported by a National Institutes of Health training grant. [H.Y.Y. was a] Research associate of the Howard Hughes Medical Institute.

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