Xenopus cyclin E, a nuclear phosphoprotein, accumulates when oocytes gain the ability to initiate DNA replication

1996 ◽  
Vol 109 (6) ◽  
pp. 1173-1184 ◽  
Author(s):  
S. Chevalier ◽  
A. Couturier ◽  
I. Chartrain ◽  
R. Le Guellec ◽  
C. Beckhelling ◽  
...  
Keyword(s):  
Dna Replication ◽  
Oocyte Maturation ◽  
Cyclin E ◽  
Amino Acid Identity ◽  
S Phase ◽  
Interphase Nuclei ◽  
Initiation Of Replication ◽  
Egg Extracts ◽  
Polysomal Fraction ◽  
Mitotic Chromatin

The capacity to initiate DNA replication appears during oocyte maturation in Xenopus. Initiation of S phase is driven by several components which include active cyclin/cdk complexes. We have identified three Xenopus cyclin E clones showing 59% amino acid identity with human cyclin E. The recruitment of cyclin E mRNA, like cdk2 mRNA, into the polysomal fraction during oocyte maturation, results in the accumulation of the corresponding proteins in unfertilized eggs. Cyclin E mRNA remains polyadenylated during cleavage and anti-cyclin E antibodies detect Xlcyclin E in embryonic nuclei at this time. Cdk2 protein is necessary for the phosphorylation of radiolabelled cyclin E added to egg extracts. Radiolabelled Xlcyclin E enters interphase nuclei and, though stable through interphase and mitosis, is not associated with condensed mitotic chromatin. In egg extracts, endogenous Xlcyclin E rapidly associates with nuclei before S phase and remains nuclear throughout interphase, becoming nucleoplasmic in G2/prophase. Under conditions where initiation of replication is limiting in extracts, Xlcyclin E associates only with those nuclei that undergo S phase. These features are entirely consistent with the view that Xlcyclin E is required for initiation of S phase.

scholarly journals Deregulation of cyclin E in human cells interferes with prereplication complex assembly

2004 ◽  
Vol 165 (6) ◽  
pp. 789-800 ◽  
Author(s):  
Susanna Ekholm-Reed ◽  
Juan Méndez ◽  
Donato Tedesco ◽  
Anders Zetterberg ◽  
Bruce Stillman ◽  
...  
Keyword(s):  
Dna Replication ◽  
Cell Lines ◽  
Broad Spectrum ◽  
Cyclin E ◽  
Chromosome Instability ◽  
S Phase ◽  
Potential Mechanism ◽  
Minichromosome Maintenance ◽  
Origins Of Replication ◽  
Initiation Of Replication

Deregulation of cyclin E expression has been associated with a broad spectrum of human malignancies. Analysis of DNA replication in cells constitutively expressing cyclin E at levels similar to those observed in a subset of tumor-derived cell lines indicates that initiation of replication and possibly fork movement are severely impaired. Such cells show a specific defect in loading of initiator proteins Mcm4, Mcm7, and to a lesser degree, Mcm2 onto chromatin during telophase and early G1 when Mcm2–7 are normally recruited to license origins of replication. Because minichromosome maintenance complex proteins are thought to function as a heterohexamer, loading of Mcm2-, Mcm4-, and Mcm7-depleted complexes is likely to underlie the S phase defects observed in cyclin E–deregulated cells, consistent with a role for minichromosome maintenance complex proteins in initiation of replication and fork movement. Cyclin E–mediated impairment of DNA replication provides a potential mechanism for chromosome instability observed as a consequence of cyclin E deregulation.

scholarly journals Both cyclin A and cyclin E have S-phase promoting (SPF) activity in Xenopus egg extracts

1996 ◽  
Vol 109 (6) ◽  
pp. 1555-1563 ◽  
Author(s):  
U.P. Strausfeld ◽  
M. Howell ◽  
P. Descombes ◽  
S. Chevalier ◽  
R.E. Rempel ◽  
...  
Keyword(s):  
Dna Replication ◽  
Cyclin E ◽  
Cyclin A ◽  
Early Embryo ◽  
S Phase ◽  
Chromosomal Dna ◽  
Cyclin E1 ◽  
Egg Extracts ◽  
High Concentrations ◽  
Xenopus Egg Extracts

Extracts of activated Xenopus eggs in which protein synthesis has been inhibited support a single round of chromosomal DNA replication. Affinity-depletion of cyclin dependent kinases (Cdks) from these extracts blocks the initiation of DNA replication. We define ‘S-phase promoting factor’ (SPF) as the Cdk activity required for DNA replication in these Cdk-depleted extracts. Recombinant cyclins A and E, but not cyclin B, showed significant SPF activity. High concentrations of cyclin A promoted entry into mitosis, which inhibited DNA replication. In contrast, high concentrations of cyclin E1 promoted neither nuclear envelope disassembly nor full chromosome condensation. In the early embryo cyclin E1 complexes exclusively with Cdk2 and cyclin A is complexed predominantly with Cdc2; only later in development does cyclin A associate with Cdk2. We show that baculovirus-produced complexes of cyclin A-Cd2, cyclin A-Cdk2 and cyclin E-Cdk2 could each provide SPF activity. These results suggest that although in the early Xenopus embryo cyclin E1-Cdk2 is sufficient to support entry into S-phase, cyclin A-Cdc2 provides a significant additional quantity of SPF as its levels rise during S phase.

scholarly journals DNA replication of mitotic chromatin in Xenopus egg extracts

2003 ◽  
Vol 100 (23) ◽  
pp. 13241-13246 ◽  
Author(s):  
T. A. Prokhorova ◽  
K. Mowrer ◽  
C. H. Gilbert ◽  
J. C. Walter
Keyword(s):  
Dna Replication ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts ◽  
Mitotic Chromatin

scholarly journals Determination of initiation of DNA replication before and after nuclear formation in Xenopus egg cell free extracts.

1993 ◽  
Vol 123 (6) ◽  
pp. 1321-1331 ◽  
Author(s):  
Y Kubota ◽  
H Takisawa
Keyword(s):  
Dna Replication ◽  
Specific Activity ◽  
S Phase ◽  
Egg Cell ◽  
Sperm Dna ◽  
Before And After ◽  
M Phase ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Initiation Of Dna Replication

Xenopus egg extracts prepared before and after egg activation retain M- and S-phase specific activity, respectively. Staurosporine, a potent inhibitor of protein kinase, converted M-phase extracts into interphase-like extracts that were capable of forming nuclei upon the addition of sperm DNA. The nuclei formed in the staurosporine treated M-phase extract were incapable of replicating DNA, and they were unable to initiate replication upon the addition of S-phase extracts. Furthermore, replication was inhibited when the staurosporine-treated M-phase extract was added in excess to the staurosporine-treated S-phase extract before the addition of DNA. The membrane-depleted S-phase extract supported neither nuclear formation nor replication; however, preincubation of sperm DNA with these extracts allowed them to form replication-competent nuclei upon the addition of excess staurosporine-treated M-phase extract. These results demonstrate that positive factors in the S-phase extracts determined the initiation of DNA replication before nuclear formation, although these factors were unable to initiate replication after nuclear formation.

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

1997 ◽  
Vol 137 (1) ◽  
pp. 183-192 ◽  
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.

scholarly journals Mutations of the Drosophila dDP,dE2F, and cyclin E Genes Reveal Distinct Roles for the E2F-DP Transcription Factor and Cyclin E during the G1-S Transition

1998 ◽  
Vol 18 (1) ◽  
pp. 141-151 ◽  
Author(s):  
Robert J. Duronio ◽  
Peter C. Bonnette ◽  
Patrick H. O’Farrell
Keyword(s):  
Dna Replication ◽  
Transcriptional Activation ◽  
Cyclin E ◽  
S Phase ◽  
Brdu Incorporation ◽  
Gene Products ◽  
Hypomorphic Allele ◽  
Transition Mutation ◽  
Replication Factors ◽  
Phase Entry

ABSTRACT Activation of heterodimeric E2F-DP transcription factors can drive the G1-S transition. Mutation of the Drosophila melanogaster dE2F gene eliminates transcriptional activation of several replication factors at the G1-S transition and compromises DNA replication. Here we describe a mutation in theDrosophila dDP gene. As expected for a defect in the dE2F partner, this mutation blocks G1-S transcription ofDmRNR2 and cyclin E as previously described for mutations of dE2F. Mutation of dDP also causes an incomplete block of DNA replication. When S phase is compromised by reducing the activity of dE2F-dDP by either a dE2F ordDP mutation, the first phenotype detected is a reduction in the intensity of BrdU incorporation and a prolongation of the labeling. Notably, in many cells, there was no detected delay in entry into this compromised S phase. In contrast, when cyclin E function was reduced by a hypomorphic allele combination, BrdU incorporation was robust but the timing of S-phase entry was delayed. We suggest that dE2F-dDP contributes to the expression of two classes of gene products: replication factors, whose abundance has a graded effect on replication, and cyclin E, which triggers an all-or-nothing transition from G1 to S phase.

scholarly journals Direct regulation of Treslin by cyclin-dependent kinase is essential for the onset of DNA replication

2011 ◽  
Vol 193 (6) ◽  
pp. 995-1007 ◽  
Author(s):  
Akiko Kumagai ◽  
Anna Shevchenko ◽  
Andrej Shevchenko ◽  
William G. Dunphy
Keyword(s):  
Dna Replication ◽  
Deoxyribonucleic Acid ◽  
S Phase ◽  
Human Cells ◽  
Target Sequence ◽  
Cyclin Dependent Kinase ◽  
Functional Importance ◽  
Interacting Protein ◽  
Egg Extracts

Treslin, a TopBP1-interacting protein, is necessary for deoxyribonucleic acid (DNA) replication in vertebrates. Association between Treslin and TopBP1 requires cyclin-dependent kinase (Cdk) activity in Xenopus laevis egg extracts. We investigated the mechanism and functional importance of Cdk for this interaction using both X. laevis egg extracts and human cells. We found that Treslin also associated with TopBP1 in a Cdk-regulated manner in human cells and that Treslin was phosphorylated within a conserved Cdk consensus target sequence (on S976 in X. laevis and S1000 in humans). Recombinant human Cdk2–cyclin E also phosphorylated this residue of Treslin in vitro very effectively. Moreover, a mutant of Treslin that cannot undergo phosphorylation on this site showed significantly diminished binding to TopBP1. Finally, human cells harboring this mutant were severely deficient in DNA replication. Collectively, these results indicate that Cdk-mediated phosphorylation of Treslin during S phase is necessary for both its effective association with TopBP1 and its ability to promote DNA replication in human cells.

scholarly journals p21cip1 Degradation in Differentiated Keratinocytes Is Abrogated by Costabilization with Cyclin E Induced by Human Papillomavirus E7

2001 ◽  
Vol 75 (13) ◽  
pp. 6121-6134 ◽  
Author(s):  
Francisco Noya ◽  
Wei-Ming Chien ◽  
Thomas R. Broker ◽  
Louise T. Chow
Keyword(s):  
Human Papillomavirus ◽  
Dna Replication ◽  
Cyclin E ◽  
Human Keratinocytes ◽  
S Phase ◽  
Brdu Incorporation ◽  
Primary Human Keratinocytes ◽  
Differentiated Cells ◽  
Hpv E7 ◽  
Cyclin E2

ABSTRACT The human papillomavirus (HPV) E7 protein promotes S-phase reentry in a fraction of postmitotic, differentiated keratinocytes. Here we report that these cells contain an inherent mechanism that opposes E7-induced DNA replication. In organotypic raft cultures of primary human keratinocytes, neither cyclin E nor p21cip1 is detectable in situ. However, E7-transduced differentiated cells not in S phase accumulate abundant cyclin E and p21cip1. We show that normally p21cip1 protein is rapidly degraded by proteasomes. In the presence of E7 or E6/E7, p21cip1, cyclin E, and cyclin E2 proteins were all up-regulated. The accumulation of p21cip1 protein is a posttranscriptional event, and ectopic cyclin E expression was sufficient to trigger it. In constract, cdk2 and p27kip1 were abundant in normal differentiated cells and were not significantly affected by E7. Cyclin E, cdk2, and p21cip1 or p27kip1 formed complexes, and relatively little kinase activity was found associated with cyclin E or cdk2. In patient papillomas and E7 raft cultures, all p27kip1-positive cells were negative for bromodeoxyuridine (BrdU) incorporation, but only some also contained cyclin E and p21cip1. In contrast, all cyclin E-positive cells also contained p27kip1. When the expression of p21cip1 was reduced by rottlerin, a PKC δ inhibitor, p27kip1- and BrdU-positive cells remained unchanged. These observations show that high levels of endogenous p27kip1 can prevent E7-induced S-phase reentry. This inhibition then leads to the stabilization of cyclin E and p21cip1. Since efficient initiation of viral DNA replication requires cyclin E and cdk2, its inhibition accounts for heterogeneous viral activities in productively infected lesions.

scholarly journals Mechanism of replication-coupled DNA-protein crosslink proteolysis by SPRTN and the proteasome

2018 ◽  
Author(s):  
Alan Gao ◽  
Nicolai B. Larsen ◽  
Justin L. Sparks ◽  
Irene Gallina ◽  
Matthias Mann ◽  
...  
Keyword(s):  
Dna Replication ◽  
S Phase ◽  
Dna Lesions ◽  
List Type ◽  
Genomic Integrity ◽  
Dna Metabolism ◽  
Single Stranded Dna ◽  
Egg Extracts ◽  
Protein Crosslinks ◽  
Bulky Dna Lesions

SummaryDNA-protein crosslinks (DPCs) are bulky DNA lesions that interfere with DNA metabolism and therefore threaten genomic integrity. Recent studies implicate the metalloprotease SPRTN in S-phase removal of DPCs, but how SPRTN activity is coupled to DNA replication is unknown. Using Xenopus egg extracts that recapitulate replication-coupled DPC proteolysis, we show that DPCs can be degraded by SPRTN or the proteasome, which act as independent DPC proteases. Proteasome recruitment requires DPC polyubiquitylation, which is triggered by single-stranded DNA, a byproduct of DNA replication. In contrast, SPRTN-mediated DPC degradation is independent of DPC polyubiquitylation but requires polymerase extension of a nascent strand to the lesion. Thus, SPRTN and proteasome activities are coupled to DNA replication by distinct mechanisms and together promote replication across immovable protein barriers.HighlightsThe proteasome, in addition to SPRTN, degrades DPCs during DNA replicationProteasome-dependent DPC degradation requires DPC ubiquitylationDPC ubiquitylation is triggered by ssDNA and does not require the replisomeSPRTN-dependent DPC degradation is a post-replicative process

scholarly journals Xenopus Cdc7 function is dependent on licensing but not on XORC, XCdc6, or CDK activity and is required for XCdc45 loading

2000 ◽  
Vol 14 (12) ◽  
pp. 1528-1540
Author(s):  
Pedro Jares ◽  
J. Julian Blow
Keyword(s):  
Dna Replication ◽  
Origin Recognition Complex ◽  
Protein Complexes ◽  
Present Report ◽  
S Phase ◽  
Chromosomal Dna ◽  
Minichromosome Maintenance ◽  
Initiation Of Replication ◽  
Sequential Binding

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.

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