Cyclin/Cdk-Dependent Initiation of DNA Replication in a Human Cell-Free System

Krude, Torsten; Jackman, Mark; Pines, Jonathon; Laskey, Ronald A.

doi:10.1016/s0092-8674(00)81863-2

Cited by 305 publications

(375 citation statements)

References 48 publications

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“…The DNA helicase activity may be crucial for the initiation as well as the elongation of DNA replication. MCM proteins are not located in the replication forks, since they are not co-localized with either replication proteins of proliferating cell nuclear antigen and replication protein A (human multisubunit single-stranded DNA-binding protein) or newly replicated DNA (18,20,24,34). In addition, MCM proteins accumulate in chromatin at the G 1 phase and detach from it as the DNA replication proceeds (18,20,24,35).…”

Section: Fig 3 Protein Cross-linking Of MCM Proteins a The Pooledmentioning

confidence: 99%

A DNA Helicase Activity Is Associated with an MCM4, -6, and -7 Protein Complex

Ishimi

1997

Journal of Biological Chemistry

506

508

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All six minichromosome maintenance (MCM) proteins have DNA-dependent ATPase motifs in the central domain which is conserved from yeast to mammals. Our group purified MCM protein complexes consisting of MCM2, -4 (Cdc21), -6 (Mis5), and -7 (CDC47) proteins from HeLa cells by using histone-Sepharose column chromatography (Ishimi, Y., Ichinose, S., Omori, A., Sato K., and Kimura, H. (1996) J. Biol. Chem. 271, 24115-24122). The present study revealed that both ATPase activity and DNA helicase activity that displaces oligonucleotides annealed to single-stranded circular DNA are associated with an MCM protein complex. Both ATPase and DNA helicase activities were co-purified with a 600-kDa protein complex that is consisted of equal amounts of MCM4, -6, and -7 proteins. An immunodepletion of the MCM protein complex from the purified fraction using anti-MCM4 antibody resulted in the severe reduction of the DNA helicase activity. Displacement of DNA fragments by the DNA helicase suggested that it migrated along single-stranded DNA in the 3 to 5 direction, and the DNA helicase activity was detected only in the presence of hydrolyzable ATP or dATP. These results suggest that this helicase may be involved in the initiation of DNA replication as a DNA unwinding enzyme.There are at least six MCM 1 proteins that play an essential role in eukaryotic DNA replication as follows: MCM2, -3, -4 (Cdc21), -5, -6 (Mis5), and -7 (CDC47) proteins (see reviews in Refs. 1-3). Genetic evidence suggests that these proteins are required for the initiation of DNA replication in yeast (4, 5). The inactivation of human MCM2 protein with antibody consistently inhibits the initiation of DNA replication (6). Interaction between the MCM proteins has been reported in yeast (4, 7), and the protein complexes containing MCM proteins are detected in Drosophila (8), Xenopus (9), mouse (10, 11), and human cells (12)(13)(14). In human cells, MCM4, -6, and -7 proteins form a stable complex, and MCM2 is loosely associated with this complex (12,15,16). MCM3 and -5 proteins also form a stable complex (10, 13). All six of the MCM proteins contain DNA-dependent ATPase motifs in a central domain that is conserved from yeast to mammals, and these motifs are found in several enzymes that unwind the DNA duplex (17). These findings suggest that MCM protein complexes may be involved in the initiation of DNA replication as an enzyme that unwinds DNA in the origin region. However, DNA helicase activity of MCM proteins has not been reported.MCM proteins bind with chromatin before the initiation of DNA replication and are then released from the chromatin as DNA replication proceeds (18 -20). Our group found that MCM protein complexes containing MCM2, -4, -6, and -7 proteins bound tightly with histone H3 in vitro and were purified to near homogeneity by histone-Sepharose column chromatography (21). In the present study, it is shown that both ATPase and DNA helicase activities are associated with an MCM4, -6, and -7 protein complex. EXPERIMENTAL PROCEDURESPurification of MCM ...

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Section: Fig 3 Protein Cross-linking Of MCM Proteins a The Pooledmentioning

confidence: 99%

A DNA Helicase Activity Is Associated with an MCM4, -6, and -7 Protein Complex

Ishimi

1997

Journal of Biological Chemistry

506

508

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“…In Vitro DNA Synthesis Assay-DNA replication reactions containing 30 l of elongation buffer (60 mM KCl, 15 mM Tris-HCl, pH 7.4, 15 mM NaCl, 1 mM ␤-mercaptoethanol, 0.5 mM spermine tetrahydrochloride, 0.5 mM spermidine trihydrochloride), a buffered mixture of NTPs, dNTPs, and an energy regeneration system (yielding a final concentration of 40 mM K-HEPES, pH 7.8, 7 mM MgCl 2 , 3 mM ATP, 0.1 mM each of GTP, CTP, UTP, 0.1 mM each of dATP, dGTP, and dCTP, 0.25 M biotin-16-dUTP, 0.5 mM dithiothreitol, 40 mM creatine phosphate, and 5 g of phosphocreatine kinase) and 1 ϫ 10 5 nuclei were performed as previously described (28,29). For detection by immunofluorescence, the nucleotide mixture was supplemented with a final concentration of 0.25 M biotin-16-dUTP (Roche Diagnostics).…”

Section: Cellmentioning

confidence: 99%

Loss of Proliferative Capacity and Induction of Senescence in Oxidatively Stressed Human Fibroblasts

Chen

Stoeber

Kingsbury

et al. 2004

Journal of Biological Chemistry

136

114

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Cellular senescence can result from short, dysfunctional telomeres, oxidative stress, or oncogene expression, and may contribute to aging. To investigate the role of cellular senescence in aging it is necessary to define the time-dependent molecular events by which it is characterized. Here we investigated changes in levels of key proteins involved in cell cycle regulation, DNA replication, and stress resistance in senescing human fibroblasts following oxidative stress. An immediate response in stressed cells was dephosphorylation of retinoblastoma (Rb) and cessation of DNA synthesis. This was followed by sequential induction of p53, p21, and p16. Increase in hypophosphorylated Rb and induction of p53 and p21 by a single stress treatment was transient, whereas sustained induction or dephosphorylation were achieved by a second stress. Down-regulation of the critical DNA replication initiation factor Cdc6 occurred early after stress concurring with p53 induction, and was followed by a decrease in Mcm2 levels. A late event in the stress-induced molecular sequence was the induction of SOD1, catalase, and HSP27 coinciding with development of the fully senescent phenotype. Our data suggest that loss of proliferative capacity in oxidatively stressed cells is a multistep process regulated by time-dependent molecular events that may play differential roles in induction and maintenance of cellular senescence.

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“…In vitro data in mammalian cells suggest that nuclear but not cytosolic cyclin E and Cdk2 are required for initiation of DNA synthesis. 25,26 We therefore concentrated our analysis mainly on nuclear expression of both proteins. No major changes of both proteins were found in the cytosol (not shown).…”

Section: Cyclin E and Cdk2 Nuclear Redistribution And Upregulation Ismentioning

confidence: 99%

Blunted DNA synthesis and delayed S-phase entry following inhibition of Cdk2 activity in the regenerating rat liver

Stärkel

Saeger

Sempoux

et al. 2005

Laboratory Investigation

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Activation of the cyclin E/Cdk2 complex may play an important role in mid-G1/S-phase progression in proliferating mammalian cells. We evaluated the effect of targeted inhibition of Cdk2 activity by CYC202 (Rroscovitine) on hepatocytes proliferation in vivo after 70% partial hepatectomy (PH) in rats. In controls, Cdk2 activity and DNA synthesis peaked 24 h after PH. CYC202 abrogated Cdk2 activity, prevented BrdU incorporation and PCNA expression and increased mortality 24 h after PH. Cyclin E and Cdk2 protein expression and complex formation was not affected by CYC202 nor was cyclin D1, Cdk4 and c-ras mRNA expression. Two consecutive injections 8 and 20 h after PH were required to elicit the inhibitory effect of CYC202, which was lost when either the injection at 8 h or at 20 h was withheld. Cdk2 activity and cell progression resumed 48 h after PH in surviving animals suggesting that CYC202 induced a reversible inhibition of the cell cycle. Our results confirm an important role for Cdk2 in hepatocytes proliferation in the regenerating liver. We demonstrate that molecular events, including Cdk2 activation, occurring within the 8th and 24th hour after PH (G1/S-phase transition) are crucial in determining whether or not DNA synthesis and hepatocytes proliferation proceed normally after PH.

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Cyclin/Cdk-Dependent Initiation of DNA Replication in a Human Cell-Free System

Cited by 305 publications

References 48 publications

A DNA Helicase Activity Is Associated with an MCM4, -6, and -7 Protein Complex

A DNA Helicase Activity Is Associated with an MCM4, -6, and -7 Protein Complex

Loss of Proliferative Capacity and Induction of Senescence in Oxidatively Stressed Human Fibroblasts

Blunted DNA synthesis and delayed S-phase entry following inhibition of Cdk2 activity in the regenerating rat liver

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