Disruption of Nuclear Lamin Organization Blocks the Elongation Phase of DNA Replication

• Published in: The Journal of cell biology 2000-06, Vol.149 (6), p.1179-1191
• Main Authors: Moir, Robert D., Spann, Timothy P., Herrmann, Harald, Goldman, Robert D.
• Format: Article
• Language: English
• Subjects: Animals; AraC protein; CDC2-CDC28 Kinases; cdk2 kinase; Cell aggregates; Cell lines; Cells; Cellular biology; Chromatin; CIP protein; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinases - antagonists & inhibitors; Cyclins - pharmacology; Cytarabine - pharmacology; Deoxyribonucleic acid; DNA; DNA Replication; elongation factor; Enzyme Inhibitors - pharmacology; Fluorescent Antibody Technique; Humans; Intermediate Filament Proteins; Interphase; Lamin Type B; Lamins; Mutation; Nuclear Envelope - metabolism; Nuclear lamina; Nuclear Localization Signals; Nuclear membrane; Nuclear Proteins - genetics; Nuclear Proteins - metabolism; Oocytes; Original; Peptide Elongation Factors - metabolism; Proliferating Cell Nuclear Antigen - metabolism; Protein Serine-Threonine Kinases - antagonists & inhibitors; replication factor; Xenopus; Xenopus Proteins
• ISSN: 0021-9525; 1540-8140
• DOI: 10.1083/jcb.149.6.1179

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 NH2-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 ΔNLA. These findings demonstrate that lamin organization must be maintained in nuclei for the elongation phase of DNA replication to proceed.