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Disruption of nuclear lamin organization blocks the elongation phase of DNA replication
R D Moir1, T P Spann, H Herrmann
1Department of Cell and Molecular Biology, Northwestern University Medical School, Chicago, Illinois 60611, USA.
Abstract:
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.
Insights
Nuclear lamins are crucial for DNA replication elongation. Disrupting lamin organization inhibits DNA synthesis by affecting elongation factors, not initiation. Maintaining lamin structure is essential for successful DNA replication progression.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- The precise role of nuclear lamins in DNA replication remains largely undetermined.
- Nuclear lamins form the nuclear lamina, a structural network within the nucleus.
Purpose of the Study:
- To investigate the function of nuclear lamins during the DNA replication process.
- To determine if nuclear lamins are involved in the initiation or elongation phases of DNA replication.
Main Methods:
- Nuclei were assembled in Xenopus extracts with AraC, a reversible replication inhibitor.
- Dominant-negative lamin mutants (DeltaNLA) were used to disrupt nuclear lamin organization.
- Replication resumption was monitored after AraC release and analyzed for effects on initiation and elongation factors.
Main Results:
- Disruption of nuclear lamin organization inhibited DNA replication resumption after AraC release.
- This inhibition was specific to the elongation phase, not initiation, as replication resumed after CIP treatment.
- Lamin disruption by DeltaNLA impaired the organization of elongation factors like proliferating cell nuclear antigen and replication factor complex.
Conclusions:
- Nuclear lamin organization is essential for the elongation phase of DNA replication.
- Proper nuclear lamin structure is required for the function of DNA replication elongation factors.
- These findings highlight a critical role for nuclear lamins in maintaining genome stability during replication.