Xenopus CDC7/DRF1 Complex Is Required for the Initiation of DNA Replication

Silva, Tania; Bradley, Rachael H; Gao, Yuefeng; Coué, Martine

doi:10.1074/jbc.m510278200

Cited by 27 publications

(46 citation statements)

References 42 publications

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“…DDK interacts stably with pre-RCs (Takahashi and Walter 2005;Silva et al 2006) and with Scc2-Scc4 (this study), suggesting that DDK plays a structural role in Scc2-Scc4 recruitment. Importantly, we also showed that the protein kinase activity of DDK is required to promote Scc2-Scc4 loading onto chromatin.…”

Section: Discussionsupporting

confidence: 58%

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Cdc7–Drf1 kinase links chromosome cohesion to the initiation of DNA replication in Xenopus egg extracts

Takahashi

Basu²,

Bermudez³

et al. 2008

Genes Dev.

109

119

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To establish functional cohesion between replicated sister chromatids, cohesin is recruited to chromatin before S phase. Cohesin is loaded onto chromosomes in the G1 phase by the Scc2-Scc4 complex, but little is known about how Scc2-Scc4 itself is recruited to chromatin. Using Xenopus egg extracts as a vertebrate model system, we showed previously that the chromatin association of Scc2 and cohesin is dependent on the prior establishment of prereplication complexes (pre-RCs) at origins of replication. Here, we report that Scc2-Scc4 exists in a stable complex with the Cdc7-Drf1 protein kinase (DDK), which is known to bind pre-RCs and activate them for DNA replication. Immunodepletion of DDK from Xenopus egg extracts impairs chromatin association of Scc2-Scc4, a defect that is reversed by wild-type, but not catalytically inactive DDK. A complex of Scc4 and the N terminus of Scc2 is sufficient for chromatin loading of Scc2-Scc4, but not for cohesin recruitment. These results show that DDK is required to tether Scc2-Scc4 to pre-RCs, and they underscore the intimate link between early steps in DNA replication and cohesion.[Keywords: Cdc7-Drf1; DNA replication; Xenopus; cohesin; cohesion; Scc2-Scc4] Supplemental material is available at http://www.genesdev.org. Chromosomal DNA is duplicated in the S phase of the cell cycle and distributed to daughter cells in mitosis. Cohesion of sister chromatids is crucial for precise segregation of chromosomes, as it marks sister chromosomes to be separated. Cohesion is established by the ring-shaped cohesin complex, which consists of two structural maintenance of chromosome (SMC) family proteins, Smc1 and Smc3, an ␣-kleisin subunit, Rad21/Scc1, and an accessory protein, SA/Scc3 (for review, see Huang et al. 2005;Nasmyth and Haering 2005;Hirano 2006;Losada 2007). Several lines of evidence suggest that cohesion is established when replicated sister chromatids are both entrapped within the cohesin ring Nasmyth 2005, 2007), but alternative models also exist (Milutinovich et al. 2007). Proteolytic cleavage of Scc1 by separase at the metaphase-anaphase transition releases cohesins from chromatin, enabling sister chromatids to move to each daughter cell (Uhlmann et al. 2000). In metazoans, cohesins associated with the chromosome arms are removed in prophase by phosphorylation of the SA subunit via polo-like kinase (Sumara et al. 2000;Waizenegger et al. 2000;Losada et al. 2002;Hauf et al. 2005). The remainder of chromosome-linked cohesins is cleaved at the metaphase-anaphase transition by separase.The process of cohesion establishment is intimately connected to DNA replication. Experiments in yeast show that the establishment of cohesion in S phase requires that cohesin be present on chromatin during DNA replication (Uhlmann and Nasmyth 1998; Lengronne et al. 2006). The Ctf7/Eco1 protein, which physically and genetically interacts with PCNA and RFC (Kenna and Skibbens 2003;Moldovan et al. 2006), is required for establishment of cohesion in S phase (Skibbens et al. 1999;Toth et al. 1999)....

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Section: Discussionsupporting

confidence: 58%

“…Cdc7-Dbf4 and Cdc7-Drf1 are often referred to as DDK (Dbf4-or Drf1-dependent kinase). During Xenopus early embryogenesis, Drf1 is the predominant activator of Cdc7, while in somatic cells, Dbf4 likely plays a more prominent role (Takahashi and Walter 2005;Silva et al 2006).…”

mentioning

confidence: 99%

Cdc7–Drf1 kinase links chromosome cohesion to the initiation of DNA replication in Xenopus egg extracts

Takahashi

Basu²,

Bermudez³

et al. 2008

Genes Dev.

109

119

View full text Add to dashboard Cite

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“…Although the Cdc7 protein level is constant during the cell cycle, its kinase activity fluctuates during this period because of its binding to the regulatory subunit, Dbf4, whose level peaks at S phase (3,4). Whereas only one regulatory subunit has been identified in yeast systems, mammals and Xenopus contain an additional regulatory subunit, Drf1 (5). Both Dbf4 and Drf1 appear to be important for DNA replication (3,5).…”

mentioning

confidence: 99%

“…Whereas only one regulatory subunit has been identified in yeast systems, mammals and Xenopus contain an additional regulatory subunit, Drf1 (5). Both Dbf4 and Drf1 appear to be important for DNA replication (3,5).…”

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confidence: 99%

ATR-dependent Activation of p38 MAP Kinase Is Responsible for Apoptotic Cell Death in Cells Depleted of Cdc7

Lee

2008

Journal of Biological Chemistry

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Cdc7 is a serine/threonine kinase that plays essential roles in the initiation of eukaryotic DNA replication and checkpoint response. In previous studies, depletion of Cdc7 by small interfering RNA was shown to induce an abortive S phase that led to the cell cycle arrest in normal human fibroblasts and apoptotic cell death in various cancer cells. Here we report that stressactivated p38 MAP kinase was activated and responsible for apoptotic cell death in Cdc7-depleted HeLa cells. The activation of p38 MAP kinase in the Cdc7-depleted cells was shown to depend on ATR, a major sensor kinase for checkpoint or DNA damage responses. Only the p38 MAP kinase, and not the other stress-activated kinases such as JNK or ERK, was activated, and both caspase 8 and caspase 9 were activated for the induction of apoptosis. Activation of apoptosis in Cdc7-depleted cells was completely abolished in cells treated with small interfering RNA or an inhibitor of the p38 MAP kinase, suggesting that p38 MAP kinase activation was responsible for apoptotic cell death. Taken together, we suggest that the ATR-dependent activation of the p38 MAP kinase is a major signaling pathway that induces apoptotic cell death after depletion of Cdc7 in cancer cells.

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“…Hence, the relationship between Cdc7 and Dbf4/Drf1 is functionally analogous to the cyclin-dependent kinase/cyclin relationship (5,6). In early Xenopus embryos, Drf1 is the primary Cdc7-binding partner that drives replication (7,8). The most well established target of DDK activity is the MCM2-7 complex, which is an important constituent of the putative replicative helicase.…”

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confidence: 99%

Drf1-dependent Kinase Interacts with Claspin through a Conserved Protein Motif

Gold

Dunphy

2010

Journal of Biological Chemistry

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The Dbf4/Drf1-dependent kinase (DDK) is required for the initiation of DNA replication in eukaryotes. Another protein, Claspin, mediates the activation of a cellular checkpoint response to stalled replication forks and is also a regulator of replication. In this study, we found that DDK phosphorylates Claspin in vitro and forms a nuclear complex containing Cdc7, Drf1, and Claspin in Xenopus egg extracts. In addition, purified Claspin and DDK are capable of a direct in vitro interaction. We identified a conserved binding site on Claspin required for its interaction with DDK. This site corresponds to the first of two sequence repeats in the Chk1-binding domain of Claspin. Furthermore, we have established that two amino acids in this motif, Asp 861 and Gln 866 , are essential for the interaction between Claspin and DDK. We found that mutant forms of Claspin incapable of interacting with DDK are still able to associate with and activate Chk1 in response to DNA replication blockages. However, Claspin-depleted egg extracts that have been reconstituted with these mutants of Claspin undergo DNA replication more slowly. These findings suggest that the interaction of DDK with Claspin mediates a checkpoint-independent function of Claspin related to DNA replication.

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Xenopus CDC7/DRF1 Complex Is Required for the Initiation of DNA Replication

Cited by 27 publications

References 42 publications

Cdc7–Drf1 kinase links chromosome cohesion to the initiation of DNA replication in Xenopus egg extracts

Cdc7–Drf1 kinase links chromosome cohesion to the initiation of DNA replication in Xenopus egg extracts

ATR-dependent Activation of p38 MAP Kinase Is Responsible for Apoptotic Cell Death in Cells Depleted of Cdc7

Drf1-dependent Kinase Interacts with Claspin through a Conserved Protein Motif

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