Disruption of nuclear lamin organization blocks the elongation phase of DNA replication.
The role of nuclear lamins in DNA replication is unclear. To address this, nuclei were assembled in Xenopus extracts containing AraC, a reversible inhibitor that blocks near the onset of the elongation phase of replication. Dominant-negative lamin mutants lacking their NH(2)-terminal domains were added to assembled nuclei to disrupt lamin organization. This prevented the resumption of DNA replication after the release of the AraC block. This inhibition of replication was not due to gross disruption of nuclear envelope structure and function. The organization of initiation factors was not altered by lamin disruption, and nuclei resumed replication when transferred to extracts treated with CIP, an inhibitor of the cyclin-dependent kinase (cdk) 2-dependent step of initiation. This suggests that alteration of lamin organization does not affect the initiation phase of DNA replication. Instead, we find that disruption of lamin organization inhibited chain elongation in a dose-dependent fashion. Furthermore, the established organization of two elongation factors, proliferating cell nuclear antigen, and replication factor complex, was disrupted by DeltaNLA. These findings demonstrate that lamin organization must be maintained in nuclei for the elongation phase of DNA replication to proceed.
Moir
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Department of Cell and Molecular Biology, Northwestern University Medical School, Chicago, Illinois 60611, USA.
Spann
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Herrmann
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Goldman
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D000818:Animals; D042846:CDC2-CDC28 Kinases; D051357:Cyclin-Dependent Kinase 2; D050759:Cyclin-Dependent Kinase Inhibitor p21; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors; D016213:Cyclins / Q000494:pharmacology; D003561:Cytarabine / Q000494:pharmacology; D004261:DNA Replication; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D006801:Humans; D007381:Intermediate Filament Proteins; D034921:Lamin Type B; D034882:Lamins; D009154:Mutation; D009685:Nuclear Envelope / Q000378:metabolism; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D009865:Oocytes; D010445:Peptide Elongation Factors / Q000378:metabolism; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors; D014981:Xenopus; D029867:Xenopus Proteins
C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004791:Enzyme Inhibitors; D007381:Intermediate Filament Proteins; D034921:Lamin Type B; D034882:Lamins; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins; D010445:Peptide Elongation Factors; D018809:Proliferating Cell Nuclear Antigen; D029867:Xenopus Proteins; D003561:Cytarabine; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; C495891:Cdk2 protein, Xenopus; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases