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© The Rockefeller University Press, 0021-9525/2000//1179 $5.00
The Journal of Cell Biology, Volume 149, Number 6, , 2000 1179-1192

Original Article

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



Robert D. Moira, Timothy P. Spanna, Harald Herrmannb, and Robert D. Goldmana

a Department of Cell and Molecular Biology, Northwestern University Medical School, Chicago, Illinois 60611
b Division of Cell Biology, German Cancer Research Center, D-69120 Heidelberg, Germany
Department of Cell and Molecular Biology, Northwestern University Medical School, 303 E. Chicago Ave., Ward 11-145, Chicago, IL 60611.(312) 503-0954(312) 503-4215

r-goldman{at}nwu.edu

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 {Delta}NLA. These findings demonstrate that lamin organization must be maintained in nuclei for the elongation phase of DNA replication to proceed.

Key Words: DNA synthesis • lamina • intermediate filaments • nuclear organization • lamin mutant

© 2000 The Rockefeller University Press

T.P. Spann and R.D. Moir contributed equally to this work.

Abbreviations used in this paper: cdk, cyclin-dependent kinase; GST, glutathione S-transferase; LA, human nuclear lamin A; LB3, Xenopus nuclear lamin B3; NLS, nuclear localization signal; NWB, nuclear wash buffer; PCNA, proliferating cell nuclear antigen; RFC, replication factor complex; XMCM, Xenopus minichromosome maintenance; factor XORC2, Xenopus origin replication complex factor 2.


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