XB-ART-16643
J Cell Biol
April 7, 1997;
137
(1):
183-92.
A role for Cdk2 kinase in negatively regulating DNA replication during S phase of the cell cycle.
Hua XH
,
Yan H
,
Newport J
.
Abstract
Using cell-free extracts made from Xenopus eggs, we show that cdk2-cyclin E and A kinases play an important role in negatively regulating DNA replication. Specifically, we demonstrate that the cdk2 kinase concentration surrounding chromatin in extracts increases 200-fold once the chromatin is assembled into nuclei. Further, we find that if the cdk2-cyclin E or A concentration in egg cytosol is increased 16-fold before the addition of sperm chromatin, the chromatin fails to initiate DNA replication once assembled into nuclei. This demonstrates that cdk2-cyclin E or A can negatively regulate DNA replication. With respect to how this negative regulation occurs, we show that high levels of cdk2-cyclin E do not block the association of the protein complex ORC with sperm chromatin but do prevent association of MCM3, a protein essential for replication. Importantly, we find that MCM3 that is prebound to chromatin does not dissociate when cdk2-cyclin E levels are increased. Taken together our results strongly suggest that during the embryonic cell cycle, the low concentrations of cdk2-cyclin E present in the cytosol after mitosis and before nuclear formation allow proteins essential for potentiating DNA replication to bind to chromatin, and that the high concentration of cdk2-cyclin E within nuclei prevents MCM from reassociating with chromatin after replication. This situation could serve, in part, to limit DNA replication to a single round per cell cycle.
PubMed ID: 9105046
PMC ID: PMC2139856
Article link:
Grant support: [+]
GM44656 NIGMS NIH HHS
Species referenced: Xenopus
Genes referenced: cdk2 mcm3 mmut orc2
Article Images: [+] show captions
References [+] :
Bell,
Yeast origin recognition complex functions in transcription silencing and DNA replication.
1994, Pubmed
Bell,
Yeast origin recognition complex functions in transcription silencing and DNA replication.
1994,
Pubmed
Blow, A role for the nuclear envelope in controlling DNA replication within the cell cycle. 1988, Pubmed , Xenbase
Blow, Preventing re-replication of DNA in a single cell cycle: evidence for a replication licensing factor. 1993, Pubmed , Xenbase
Blow, Initiation of DNA replication in nuclei and purified DNA by a cell-free extract of Xenopus eggs. 1986, Pubmed , Xenbase
Broek, Involvement of p34cdc2 in establishing the dependency of S phase on mitosis. 1991, Pubmed
Bueno, Dual functions of CDC6: a yeast protein required for DNA replication also inhibits nuclear division. 1992, Pubmed
Carpenter, Role for a Xenopus Orc2-related protein in controlling DNA replication. 1996, Pubmed , Xenbase
Chong, Purification of an MCM-containing complex as a component of the DNA replication licensing system. 1995, Pubmed , Xenbase
Coleman, The Xenopus Cdc6 protein is essential for the initiation of a single round of DNA replication in cell-free extracts. 1996, Pubmed , Xenbase
Correa-Bordes, p25rum1 orders S phase and mitosis by acting as an inhibitor of the p34cdc2 mitotic kinase. 1996, Pubmed
Dahmann, S-phase-promoting cyclin-dependent kinases prevent re-replication by inhibiting the transition of replication origins to a pre-replicative state. 1996, Pubmed
Dalton, Cell cycle-regulated nuclear import and export of Cdc47, a protein essential for initiation of DNA replication in budding yeast. 1995, Pubmed
Donovan, Replication origins in eukaroytes. 1996, Pubmed , Xenbase
Dulić, Association of human cyclin E with a periodic G1-S phase protein kinase. 1992, Pubmed
Dunphy, The Xenopus cdc2 protein is a component of MPF, a cytoplasmic regulator of mitosis. 1988, Pubmed , Xenbase
Fang, Evidence that the G1-S and G2-M transitions are controlled by different cdc2 proteins in higher eukaryotes. 1991, Pubmed , Xenbase
Gavin, Conserved initiator proteins in eukaryotes. 1996, Pubmed
Guadagno, Cdk2 kinase is required for entry into mitosis as a positive regulator of Cdc2-cyclin B kinase activity. 1996, Pubmed , Xenbase
Hayles, Temporal order of S phase and mitosis in fission yeast is determined by the state of the p34cdc2-mitotic B cyclin complex. 1994, Pubmed
Hennessy, A group of interacting yeast DNA replication genes. 1991, Pubmed
Howe, A developmental timer regulates degradation of cyclin E1 at the midblastula transition during Xenopus embryogenesis. 1996, Pubmed , Xenbase
Jackson, Early events in DNA replication require cyclin E and are blocked by p21CIP1. 1995, Pubmed , Xenbase
Kelly, The fission yeast cdc18+ gene product couples S phase to START and mitosis. 1993, Pubmed
Koff, Formation and activation of a cyclin E-cdk2 complex during the G1 phase of the human cell cycle. 1992, Pubmed
Kornbluth, In vitro cell cycle arrest induced by using artificial DNA templates. 1992, Pubmed , Xenbase
Kubota, Identification of the yeast MCM3-related protein as a component of Xenopus DNA replication licensing factor. 1995, Pubmed , Xenbase
Kubota, Determination of initiation of DNA replication before and after nuclear formation in Xenopus egg cell free extracts. 1994, Pubmed , Xenbase
Leno, The nuclear membrane prevents replication of human G2 nuclei but not G1 nuclei in Xenopus egg extract. 1992, Pubmed , Xenbase
Liang, ORC and Cdc6p interact and determine the frequency of initiation of DNA replication in the genome. 1995, Pubmed
Lohka, Roles of cytosol and cytoplasmic particles in nuclear envelope assembly and sperm pronuclear formation in cell-free preparations from amphibian eggs. 1984, Pubmed , Xenbase
Madine, The nuclear envelope prevents reinitiation of replication by regulating the binding of MCM3 to chromatin in Xenopus egg extracts. 1996, Pubmed , Xenbase
Madine, MCM3 complex required for cell cycle regulation of DNA replication in vertebrate cells. 1995, Pubmed , Xenbase
Moreno, Regulation of progression through the G1 phase of the cell cycle by the rum1+ gene. 1994, Pubmed
Newport, Nuclear reconstitution in vitro: stages of assembly around protein-free DNA. 1987, Pubmed , Xenbase
Nishitani, p65cdc18 plays a major role controlling the initiation of DNA replication in fission yeast. 1996, Pubmed
Piatti, Activation of S-phase-promoting CDKs in late G1 defines a "point of no return" after which Cdc6 synthesis cannot promote DNA replication in yeast. 1996, Pubmed
Piatti, 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, Pubmed
Pines, Human cyclins A and B1 are differentially located in the cell and undergo cell cycle-dependent nuclear transport. 1991, Pubmed
Rao, The origin recognition complex interacts with a bipartite DNA binding site within yeast replicators. 1995, Pubmed
Rempel, Maternal Xenopus Cdk2-cyclin E complexes function during meiotic and early embryonic cell cycles that lack a G1 phase. 1995, Pubmed , Xenbase
Sauer, Distinct modes of cyclin E/cdc2c kinase regulation and S-phase control in mitotic and endoreduplication cycles of Drosophila embryogenesis. 1995, Pubmed
Solomon, Cyclin activation of p34cdc2. 1991, Pubmed , Xenbase
Strausfeld, Cip1 blocks the initiation of DNA replication in Xenopus extracts by inhibition of cyclin-dependent kinases. 1995, Pubmed , Xenbase
Su, Qualifying for the license to replicate. 1995, Pubmed , Xenbase
Todorov, BM28, a human member of the MCM2-3-5 family, is displaced from chromatin during DNA replication. 1995, Pubmed
Tye, The MCM2-3-5 proteins: are they replication licensing factors? 2004, Pubmed
Usui, Uncoupled cell cycle without mitosis induced by a protein kinase inhibitor, K-252a. 1991, Pubmed
VanRenterghem, Regulation of mitogen-activated protein kinase activation by protein kinases A and C in a cell-free system. 1994, Pubmed , Xenbase
Yan, Cell cycle-regulated nuclear localization of MCM2 and MCM3, which are required for the initiation of DNA synthesis at chromosomal replication origins in yeast. 1993, Pubmed
Yan, An analysis of the regulation of DNA synthesis by cdk2, Cip1, and licensing factor. 1995, Pubmed , Xenbase
Yan, Mcm2 and Mcm3, two proteins important for ARS activity, are related in structure and function. 1991, Pubmed
Blow, A role for the nuclear envelope in controlling DNA replication within the cell cycle. 1988, Pubmed , Xenbase
Blow, Preventing re-replication of DNA in a single cell cycle: evidence for a replication licensing factor. 1993, Pubmed , Xenbase
Blow, Initiation of DNA replication in nuclei and purified DNA by a cell-free extract of Xenopus eggs. 1986, Pubmed , Xenbase
Broek, Involvement of p34cdc2 in establishing the dependency of S phase on mitosis. 1991, Pubmed
Bueno, Dual functions of CDC6: a yeast protein required for DNA replication also inhibits nuclear division. 1992, Pubmed
Carpenter, Role for a Xenopus Orc2-related protein in controlling DNA replication. 1996, Pubmed , Xenbase
Chong, Purification of an MCM-containing complex as a component of the DNA replication licensing system. 1995, Pubmed , Xenbase
Coleman, The Xenopus Cdc6 protein is essential for the initiation of a single round of DNA replication in cell-free extracts. 1996, Pubmed , Xenbase
Correa-Bordes, p25rum1 orders S phase and mitosis by acting as an inhibitor of the p34cdc2 mitotic kinase. 1996, Pubmed
Dahmann, S-phase-promoting cyclin-dependent kinases prevent re-replication by inhibiting the transition of replication origins to a pre-replicative state. 1996, Pubmed
Dalton, Cell cycle-regulated nuclear import and export of Cdc47, a protein essential for initiation of DNA replication in budding yeast. 1995, Pubmed
Donovan, Replication origins in eukaroytes. 1996, Pubmed , Xenbase
Dulić, Association of human cyclin E with a periodic G1-S phase protein kinase. 1992, Pubmed
Dunphy, The Xenopus cdc2 protein is a component of MPF, a cytoplasmic regulator of mitosis. 1988, Pubmed , Xenbase
Fang, Evidence that the G1-S and G2-M transitions are controlled by different cdc2 proteins in higher eukaryotes. 1991, Pubmed , Xenbase
Gavin, Conserved initiator proteins in eukaryotes. 1996, Pubmed
Guadagno, Cdk2 kinase is required for entry into mitosis as a positive regulator of Cdc2-cyclin B kinase activity. 1996, Pubmed , Xenbase
Hayles, Temporal order of S phase and mitosis in fission yeast is determined by the state of the p34cdc2-mitotic B cyclin complex. 1994, Pubmed
Hennessy, A group of interacting yeast DNA replication genes. 1991, Pubmed
Howe, A developmental timer regulates degradation of cyclin E1 at the midblastula transition during Xenopus embryogenesis. 1996, Pubmed , Xenbase
Jackson, Early events in DNA replication require cyclin E and are blocked by p21CIP1. 1995, Pubmed , Xenbase
Kelly, The fission yeast cdc18+ gene product couples S phase to START and mitosis. 1993, Pubmed
Koff, Formation and activation of a cyclin E-cdk2 complex during the G1 phase of the human cell cycle. 1992, Pubmed
Kornbluth, In vitro cell cycle arrest induced by using artificial DNA templates. 1992, Pubmed , Xenbase
Kubota, Identification of the yeast MCM3-related protein as a component of Xenopus DNA replication licensing factor. 1995, Pubmed , Xenbase
Kubota, Determination of initiation of DNA replication before and after nuclear formation in Xenopus egg cell free extracts. 1994, Pubmed , Xenbase
Leno, The nuclear membrane prevents replication of human G2 nuclei but not G1 nuclei in Xenopus egg extract. 1992, Pubmed , Xenbase
Liang, ORC and Cdc6p interact and determine the frequency of initiation of DNA replication in the genome. 1995, Pubmed
Lohka, Roles of cytosol and cytoplasmic particles in nuclear envelope assembly and sperm pronuclear formation in cell-free preparations from amphibian eggs. 1984, Pubmed , Xenbase
Madine, The nuclear envelope prevents reinitiation of replication by regulating the binding of MCM3 to chromatin in Xenopus egg extracts. 1996, Pubmed , Xenbase
Madine, MCM3 complex required for cell cycle regulation of DNA replication in vertebrate cells. 1995, Pubmed , Xenbase
Moreno, Regulation of progression through the G1 phase of the cell cycle by the rum1+ gene. 1994, Pubmed
Newport, Nuclear reconstitution in vitro: stages of assembly around protein-free DNA. 1987, Pubmed , Xenbase
Nishitani, p65cdc18 plays a major role controlling the initiation of DNA replication in fission yeast. 1996, Pubmed
Piatti, Activation of S-phase-promoting CDKs in late G1 defines a "point of no return" after which Cdc6 synthesis cannot promote DNA replication in yeast. 1996, Pubmed
Piatti, 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, Pubmed
Pines, Human cyclins A and B1 are differentially located in the cell and undergo cell cycle-dependent nuclear transport. 1991, Pubmed
Rao, The origin recognition complex interacts with a bipartite DNA binding site within yeast replicators. 1995, Pubmed
Rempel, Maternal Xenopus Cdk2-cyclin E complexes function during meiotic and early embryonic cell cycles that lack a G1 phase. 1995, Pubmed , Xenbase
Sauer, Distinct modes of cyclin E/cdc2c kinase regulation and S-phase control in mitotic and endoreduplication cycles of Drosophila embryogenesis. 1995, Pubmed
Solomon, Cyclin activation of p34cdc2. 1991, Pubmed , Xenbase
Strausfeld, Cip1 blocks the initiation of DNA replication in Xenopus extracts by inhibition of cyclin-dependent kinases. 1995, Pubmed , Xenbase
Su, Qualifying for the license to replicate. 1995, Pubmed , Xenbase
Todorov, BM28, a human member of the MCM2-3-5 family, is displaced from chromatin during DNA replication. 1995, Pubmed
Tye, The MCM2-3-5 proteins: are they replication licensing factors? 2004, Pubmed
Usui, Uncoupled cell cycle without mitosis induced by a protein kinase inhibitor, K-252a. 1991, Pubmed
VanRenterghem, Regulation of mitogen-activated protein kinase activation by protein kinases A and C in a cell-free system. 1994, Pubmed , Xenbase
Yan, Cell cycle-regulated nuclear localization of MCM2 and MCM3, which are required for the initiation of DNA synthesis at chromosomal replication origins in yeast. 1993, Pubmed
Yan, An analysis of the regulation of DNA synthesis by cdk2, Cip1, and licensing factor. 1995, Pubmed , Xenbase
Yan, Mcm2 and Mcm3, two proteins important for ARS activity, are related in structure and function. 1991, Pubmed