Xenopus Cdc 7 function is dependent on licensing but not on XORC , XCdc 6 , or CDK activity and is required for XCdc 45 loading

The assembly and disassembly of protein complexes at replication origins play a crucial role in the regulation of chromosomal DNA replication. The sequential binding of the origin recognition complex (ORC), Cdc6, and the minichromosome maintenance (MCM/P1) proteins produces a licensed replication origin. Before the initiation of replication can occur, each licensed origin must be acted upon by S phase-inducing CDKs and the Cdc7 protein kinase. In the present report we describe the role of Xenopus Cdc7 (XCdc7) in DNA replication using cell-free extracts of Xenopus eggs. We show that XCdc7 binds to chromatin during G1 and S phase. XCdc7 associates with chromatin only once origins have been licensed, but this association does not require the continued presence of XORC or XCdc6 once they have fulfilled their essential role in licensing. Moreover, XCdc7 is required for the subsequent CDK-dependent loading of XCdc45 but is not required for the destabilization of origins that occurs once licensing is complete. Finally, we show that CDK activity is not necessary for XCdc7 to associate with chromatin, induce MCM/P1 phosphorylation, or perform its essential replicative function. From these results we suggest a simple model for the assembly of functional initiation complexes in the Xenopus system.

[1]  J. Blow,et al.  Sequential MCM/P1 Subcomplex Assembly Is Required to Form a Heterohexamer with Replication Licensing Activity* , 2000, The Journal of Biological Chemistry.

[2]  D. McDonald,et al.  Mammalian Cdc7–Dbf4 protein kinase complex is essential for initiation of DNA replication , 1999, The EMBO journal.

[3]  B. Stillman,et al.  Cdc7p–Dbf4p kinase binds to chromatin during S phase and is regulated by both the APC and the RAD53 checkpoint pathway , 1999, The EMBO journal.

[4]  S. Elsasser,et al.  Phosphorylation controls timing of Cdc6p destruction: A biochemical analysis. , 1999, Molecular biology of the cell.

[5]  S. Gasser,et al.  A role for the Cdc7 kinase regulatory subunit Dbf4p in the formation of initiation-competent origins of replication. , 1999, Genes & development.

[6]  O. Aparicio,et al.  Differential assembly of Cdc45p and DNA polymerases at early and late origins of DNA replication. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[7]  T. Miyake,et al.  A Fission Yeast Gene,him1+/dfp1+, Encoding a Regulatory Subunit for Hsk1 Kinase, Plays Essential Roles in S-Phase Initiation as Well as in S-Phase Checkpoint Control and Recovery from DNA Damage , 1999, Molecular and Cellular Biology.

[8]  Yiqun Shellman,et al.  Cell Cycle Control of Cdc7p Kinase Activity through Regulation of Dbf4p Stability , 1999, Molecular and Cellular Biology.

[9]  J. Blow,et al.  Changes in association of the Xenopus origin recognition complex with chromatin on licensing of replication origins. , 1999, Journal of cell science.

[10]  Liang Cheng,et al.  Cell Cycle Regulation of DNA Replication Initiator Factor Dbf4p , 1999, Molecular and Cellular Biology.

[11]  B. Roberts,et al.  DNA replication in vertebrates requires a homolog of the Cdc7 protein kinase. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[12]  J. Blow,et al.  The RLF-B component of the replication licensing system is distinct from Cdc6 and functions after Cdc6 binds to chromatin , 1999, Current Biology.

[13]  J. Blow,et al.  The regulation of replication origin activation. , 1999, Current opinion in genetics & development.

[14]  Jiri Bartek,et al.  Phosphorylation of mammalian CDC6 by Cyclin A/CDK2 regulates its subcellular localization , 1999, The EMBO journal.

[15]  K. Shirahige,et al.  Regulation of DNA-replication origins during cell-cycle progression , 1998, Nature.

[16]  J. Diffley,et al.  A Mec1- and Rad53-dependent checkpoint controls late-firing origins of DNA replication , 1998, Nature.

[17]  H. Takisawa,et al.  Xenopus Cdc45‐dependent loading of DNA polymerase α onto chromatin under the control of S‐phase cdk , 1998, The EMBO journal.

[18]  T. Kelly,et al.  Purification of Hsk1, a Minichromosome Maintenance Protein Kinase from Fission Yeast* , 1998, The Journal of Biological Chemistry.

[19]  F. Cross,et al.  CLB5-dependent activation of late replication origins in S. cerevisiae. , 1998, Molecular cell.

[20]  M. Kirschner,et al.  Geminin, an Inhibitor of DNA Replication, Is Degraded during Mitosis , 1998, Cell.

[21]  R. Hollingsworth,et al.  A human homolog of the yeast CDC7 gene is overexpressed in some tumors and transformed cell lines. , 1998, Gene.

[22]  B. Stillman,et al.  Formation of a preinitiation complex by S-phase cyclin CDK-dependent loading of Cdc45p onto chromatin. , 1998, Science.

[23]  W. L. Fangman,et al.  Cdc7 is required throughout the yeast S phase to activate replication origins. , 1998, Genes & development.

[24]  J. Diffley,et al.  The Cdc7 protein kinase is required for origin firing during S phase. , 1998, Genes & development.

[25]  J. Newport,et al.  Identification of a Preinitiation Step in DNA Replication That Is Independent of Origin Recognition Complex and cdc6, but Dependent on cdk2 , 1998, The Journal of cell biology.

[26]  T. Hunter,et al.  Identification and characterization of a human protein kinase related to budding yeast Cdc7p. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[27]  M. Lei,et al.  Mcm2 is a target of regulation by Cdc7-Dbf4 during the initiation of DNA synthesis. , 1997, Genes & development.

[28]  J. Li,et al.  CDC45 is required in conjunction with CDC7/DBF4 to trigger the initiation of DNA replication. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[29]  P. Jallepalli,et al.  Regulation of the replication initiator protein p65cdc18 by CDK phosphorylation. , 1997, Genes & development.

[30]  K. Arai,et al.  Human and Xenopus cDNAs encoding budding yeast Cdc7‐related kinases: in vitro phosphorylation of MCM subunits by a putative human homologue of Cdc7 , 1997, The EMBO journal.

[31]  H. Nojima,et al.  Licensing of DNA replication by a multi‐protein complex of MCM/P1 proteins in Xenopus eggs , 1997, The EMBO journal.

[32]  J. Blow,et al.  The RLF‐M component of the replication licensing system forms complexes containing all six MCM/P1 polypeptides , 1997, The EMBO journal.

[33]  L. Drury,et al.  Cdc6p-dependent loading of Mcm proteins onto pre-replicative chromatin in budding yeast. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[34]  P. Pahl,et al.  mcm5/cdc46-bob1 bypasses the requirement for the S phase activator Cdc7p. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[35]  B. Stillman,et al.  CDC45, a novel yeast gene that functions with the origin recognition complex and Mcm proteins in initiation of DNA replication , 1997, Molecular and cellular biology.

[36]  J. Blow,et al.  Cell Cycle Regulation of the Replication Licensing System: Involvement of a Cdk-dependent Inhibitor , 1997, The Journal of cell biology.

[37]  J. Blow,et al.  Characterization of the Xenopus replication licensing system. , 1997, Methods in enzymology.

[38]  J. Diffley,et al.  Once and only once upon a time: specifying and regulating origins of DNA replication in eukaryotic cells. , 1996, Genes & development.

[39]  J. Blow,et al.  The Xenopus origin recognition complex is essential for DNA replication and MCM binding to chromatin , 1996, Current Biology.

[40]  S. Dalton,et al.  Cdc45p assembles into a complex with Cdc46p/Mcm5p, is required for minichromosome maintenance, and is essential for chromosomal DNA replication. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[41]  G. Evan,et al.  Interaction between the Origin Recognition Complex and the Replication Licensing Systemin Xenopus , 1996, Cell.

[42]  T. Coleman,et al.  The Xenopus Cdc6 Protein Is Essential for the Initiation of a Single Round of DNA Replication in Cell-Free Extracts , 1996, Cell.

[43]  T. Hunt,et al.  The crystal structure of cyclin A. , 1995, Structure.

[44]  K. Arai,et al.  hsk1+, a Schizosaccharomyces pombe gene related to Saccharomyces cerevisiae CDC7, is required for chromosomal replication. , 1995, The EMBO journal.

[45]  James P. J. Chong,et al.  Purification of an MCM-containing complex as a component of the DNA replication licensing system , 1995, Nature.

[46]  H. Nojima,et al.  Identification of the yeast MCM3-related protein as a component of xenopus DNA replication licensing factor , 1995, Cell.

[47]  R. Fotedar,et al.  Cell cycle control of DNA replication. , 1995, Progress in cell cycle research.

[48]  U. Strausfeld,et al.  Cip1 blocks the initiation of DNA replication in Xenopus extracts by inhibition of cyclin-dependent kinases , 1994, Current Biology.

[49]  J. Diffley,et al.  Interaction of Dbf4, the Cdc7 protein kinase regulatory subunit, with yeast replication origins in vivo. , 1994, Science.

[50]  J. Diffley,et al.  Two steps in the assembly of complexes at yeast replication origins in vivo , 1994, Cell.

[51]  J. Blow,et al.  Preventing re-replication of DNA in a single cell cycle: evidence for a replication licensing factor , 1993, The Journal of cell biology.

[52]  A. Jackson,et al.  Cell cycle regulation of the yeast Cdc7 protein kinase by association with the Dbf4 protein , 1993, Molecular and cellular biology.

[53]  H. Yoon,et al.  Regulation of Saccharomyces cerevisiae CDC7 function during the cell cycle. , 1993, Molecular biology of the cell.

[54]  R. Hollingsworth,et al.  Molecular genetic studies of the Cdc7 protein kinase and induced mutagenesis in yeast. , 1992, Genetics.

[55]  J. Diffley,et al.  Protein-DNA interactions at a yeast replication origin , 1992, Nature.

[56]  Bruce Stillman,et al.  ATP-dependent recognition of eukaryotic origins of DNA replication by a multiprotein complex , 1992, Nature.

[57]  M. Kirschner,et al.  The events of the midblastula transition in Xenopus are regulated by changes in the cell cycle , 1987, Cell.

[58]  K Hara,et al.  A cytoplasmic clock with the same period as the division cycle in Xenopus eggs. , 1980, Proceedings of the National Academy of Sciences of the United States of America.