Unphosphorylatable mutants of Cdc6 disrupt its nuclear export but still support DNA replication once per cell cycle.

Cdc6 is essential for eukaryotic DNA replication. We have mutated highly conserved CDK phosphorylation sites in Cdc6. Contrary to their reported phenotypes in human cells, unphosphorylatable DeltaCDK mutants fully support DNA replication in Xenopus eggs. WtCdc6 is actively exported from the nucleus, which could explain why nuclear permeabilization is required for reinitiation within one cell cycle. However, DeltaCDK mutants are retained in the nucleus, yet surprisingly they still support only one round of replication. As these highly conserved CDK sites are unnecessary for replication once per cell cycle, an alternative checkpoint role for monitoring completion of the S phase is suggested.

[1]  R. Laskey,et al.  Chromatin-bound Cdc6 persists in S and G2 phases in human cells, while soluble Cdc6 is destroyed in a cyclin A-cdk2 dependent process. , 2000, Journal of cell science.

[2]  R. Laskey,et al.  Chromatin-bound Cdc 6 persists in S and G 2 phases in human cells , while soluble Cdc 6 is destroyed in a cyclin A-cdk 2 dependent process , 2000 .

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

[4]  M. Inagaki,et al.  Cell Cycle Regulation of Human CDC6 Protein , 1999, The Journal of Biological Chemistry.

[5]  T. Hunter,et al.  Multistep regulation of DNA replication by Cdk phosphorylation of HsCdc6. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

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

[7]  K. Stoeber,et al.  Cdc6 protein causes premature entry into S phase in a mammalian cell‐free system , 1998, The EMBO journal.

[8]  J. Parvin,et al.  Human CDC6/Cdc18 Associates with Orc1 and Cyclin-cdk and Is Selectively Eliminated from the Nucleus at the Onset of S Phase , 1998, Molecular and Cellular Biology.

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

[10]  K. Nasmyth,et al.  Loading of an Mcm Protein onto DNA Replication Origins Is Regulated by Cdc6p and CDKs , 1997, Cell.

[11]  P. Jallepalli,et al.  Interaction of the S phase regulator cdc18 with cyclin-dependent kinase in fission yeast. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[12]  D. Coverley,et al.  Xenopus replication assays. , 1997, Methods in enzymology.

[13]  M. Madine,et al.  Mechanisms restricting DNA replication to once per cell cycle: the role of Cdc6p and ORC. , 1997, Trends in cell biology.

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

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

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

[17]  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.

[18]  M. Madine,et al.  Mechanisms restricting DNA replication to once per cell cycle: MCMS, pre-replicative complexes and kinases. , 1996, Trends in cell biology.

[19]  K. Nasmyth,et al.  An essential role for the Cdc6 protein in forming the pre-replicative complexes of budding yeast , 1996, Nature.

[20]  P. Nurse,et al.  p65 cdc18 Plays a major role controlling the initiation of DNA replication in fission yeast , 1995, Cell.

[21]  R. Laskey,et al.  The nuclear envelope prevents reinitiation of replication by regulating the binding of MCM3 to chromatin in Xenopus egg extracts , 1995, Current Biology.

[22]  K. Nasmyth,et al.  S-phase-promoting cyclin-dependent kinases prevent re-replication by inhibiting the transition of replication origins to a pre-replicative state , 1995, Current Biology.

[23]  K Nasmyth,et al.  Cdc6 is an unstable protein whose de novo synthesis in G1 is important for the onset of S phase and for preventing a ‘reductional’ anaphase in the budding yeast Saccharomyces cerevisiae. , 1995, The EMBO journal.

[24]  R. Laskey,et al.  MCM3 complex required for cell cycle regulation of DNA replication in vertebrate cells , 1995, Nature.

[25]  Kim Nasmyth,et al.  The B-type cyclin kinase inhibitor p40 SIC1 controls the G1 to S transition in S. cerevisiae , 1994, Cell.

[26]  D. Fujiwara,et al.  Leptomycin B targets a regulatory cascade of crm1, a fission yeast nuclear protein, involved in control of higher order chromosome structure and gene expression. , 1994, The Journal of biological chemistry.

[27]  T. Coleman,et al.  Two distinct mechanisms for negative regulation of the Wee1 protein kinase. , 1993, The EMBO journal.

[28]  R. Laskey,et al.  Reversible effects of nuclear membrane permeabilization on DNA replication: evidence for a positive licensing factor , 1993, The Journal of cell biology.

[29]  P. Russell,et al.  Dual functions of CDC6: a yeast protein required for DNA replication also inhibits nuclear division. , 1992, The EMBO journal.

[30]  J. White,et al.  Replication occurs at discrete foci spaced throughout nuclei replicating in vitro. , 1989, Journal of cell science.

[31]  J. Blow,et al.  A role for the nuclear envelope in controlling DNA replication within the cell cycle , 1988, Nature.

[32]  J. Blow,et al.  Initiation of DNA replication in nuclei and purified DNA by a cell-free extract of Xenopus eggs , 1986, Cell.