Negative Regulation of S Phase

A Role for Cdk2 Kinase in Negatively Regulating DNA Replication during S Phase of the Cell Cycle

The Journal of Cell Biology ◽

10.1083/jcb.137.1.183 ◽

1997 ◽

Vol 137 (1) ◽

pp. 183-192 ◽

Cited By ~ 98

Author(s):

Xuequn Helen Hua ◽

Hong Yan ◽

John Newport

Keyword(s):

Cell Cycle ◽

Dna Replication ◽

Cyclin E ◽

Embryonic Cell ◽

S Phase ◽

Negative Regulation ◽

Sperm Chromatin ◽

High Concentration ◽

Low Concentrations ◽

Embryonic Cell Cycle

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.

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Negative Regulation of Cdc18 DNA Replication Protein by Cdc2

Molecular Biology of the Cell ◽

10.1091/mbc.9.1.63 ◽

1998 ◽

Vol 9 (1) ◽

pp. 63-73 ◽

Cited By ~ 55

Author(s):

Antonia Lopez-Girona ◽

Odile Mondesert ◽

Janet Leatherwood ◽

Paul Russell

Keyword(s):

Cell Cycle ◽

Dna Replication ◽

Fission Yeast ◽

Phosphorylation Site ◽

Constitutive Expression ◽

S Phase ◽

Negative Regulation ◽

Chromosomal Dna

Fission yeast Cdc18, a homologue of Cdc6 in budding yeast and metazoans, is periodically expressed during the S phase and required for activation of replication origins. Cdc18 overexpression induces DNA rereplication without mitosis, as does elimination of Cdc2-Cdc13 kinase during G2 phase. These findings suggest that illegitimate activation of origins may be prevented through inhibition of Cdc18 by Cdc2. Consistent with this hypothesis, we report that Cdc18 interacts with Cdc2 in association with Cdc13 and Cig2 B-type cyclins in vivo. Cdc18 is phosphorylated by the associated Cdc2 in vitro. Mutation of a single phosphorylation site, T104A, activates Cdc18 in the rereplication assay. The cdc18-K9 mutation is suppressed by a cig2 mutation, providing genetic evidence that Cdc2-Cig2 kinase inhibits Cdc18. Moreover, constitutive expression of Cig2 prevents rereplication in cells lacking Cdc13. These findings identify Cdc18 as a key target of Cdc2-Cdc13 and Cdc2-Cig2 kinases in the mechanism that limits chromosomal DNA replication to once per cell cycle.

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Mus81, Rhp51(Rad51), and Rqh1 Form an Epistatic Pathway Required for the S-Phase DNA Damage Checkpoint

Molecular Biology of the Cell ◽

10.1091/mbc.e08-08-0798 ◽

2009 ◽

Vol 20 (3) ◽

pp. 819-833 ◽

Cited By ~ 27

Author(s):

Nicholas Willis ◽

Nicholas Rhind

Keyword(s):

Dna Damage ◽

Dna Synthesis ◽

Replication Fork ◽

Alkylating Agent ◽

S Phase ◽

Negative Regulation ◽

Dna Damage Checkpoint ◽

Methyl Methane Sulfonate ◽

Methane Sulfonate ◽

Checkpoint Signaling

The S-phase DNA damage checkpoint slows the rate of DNA synthesis in response to damage during replication. In the fission yeast Schizosaccharomyces pombe, Cds1, the S-phase-specific checkpoint effector kinase, is required for checkpoint signaling and replication slowing; upon treatment with the alkylating agent methyl methane sulfonate, cds1Δ mutants display a complete checkpoint defect. We have identified proteins downstream of Cds1 required for checkpoint-dependant slowing, including the structure-specific endonuclease Mus81 and the helicase Rqh1, which are implicated in replication fork stability and the negative regulation of recombination. Removing Rhp51, the Rad51 recombinase homologue, suppresses the slowing defect of rqh1Δ mutants, but not that of mus81Δ mutant, defining an epistatic pathway in which mus81 is epistatic to rhp51 and rhp51 is epistatic to rqh1. We propose that restraining recombination is required for the slowing of replication in response to DNA damage.

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Negative regulation of G1 and G2 by S-phase cyclins of Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.15.9.5030 ◽

1995 ◽

Vol 15 (9) ◽

pp. 5030-5042 ◽

Cited By ~ 27

Author(s):

R D Basco ◽

M D Segal ◽

S I Reed

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Cycle ◽

Dna Replication ◽

Protein Kinase ◽

Genetic Analysis ◽

S Phase ◽

Negative Regulation ◽

Cycle Phase ◽

Wild Type ◽

Mitotic Cyclin

Cell cycle progression in the budding yeast Saccharomyces cerevisiae is controlled by the Cdc28 protein kinase, which is sequentially activated by different sets of cyclins. Previous genetic analysis has revealed that two B-type cyclins, Clb5 and Clb6, have a positive role in DNA replication. In the present study, we show, in addition, that these cyclins negatively regulate G1- and G2-specific functions. The consequences of this negative regulation were most apparent in clb6 mutants, which had a shorter pre-Start G1 phase as well as a shorter G2 phase than congenic wild-type cells. As a consequence, clb6 mutants grew and proliferated more rapidly than wild-type cells. It was more difficult to assess the role of Clb5 in G1 and G2 by genetic analysis because of the extreme prolongation of S phase in clb5 mutants. Nevertheless, both Clb5 and Clb6 were shown to be responsible for down-regulation of the protein kinase activities associated with Cln2, a G1 cyclin, and Clb2, a mitotic cyclin, in vivo. These observations are consistent with the observed cell cycle phase accelerations associated with the clb6 mutant and are suggestive of similar functions for Clb5. Genetic evidence suggested that the inhibition of mitotic cyclin-dependent kinase activities was dependent on and possibly mediated through the CDC6 gene product. Thus, Clb5 and Clb6 may stabilize S phase by promoting DNA replication while inhibiting other cell cycle activities.

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