DISRUPTION OF NUCLEAR LAMIN ORGANIZATION ALTERS THE DISTRIBUTION OF REPLICATION FACTORS AND INHIBITS DNA-SYNTHESIS

The nuclear lamina is a fibrous structure that lies at the interface b etween the nuclear envelope and the nucleoplasm. The major proteins co mprising the lamina, the nuclear lamins, are also found in foci in the nucleoplasm, distinct from the peripheral lamina. The nuclear lamins have been associated with a number of processes in the nucleus, includ ing DNA replication. To further characterize the specific role of lami ns in DNA replication, we have used a truncated human lamin as a domin ant negative mutant to perturb lamin organization. This protein disrup ts the lamin organization of nuclei when microinjected into mammalian cells and also disrupts the lamin organization of in vitro assembled n uclei when added to Xenopus laevis interphase egg extracts. In both ca ses, the lamina appears to be completely absent, and instead the endog enous lamins and the mutant lamin protein are found in nucleoplasmic a ggregates. Coincident with the disruption of lamin organization, there is a dramatic reduction in DNA replication, As a consequence of this disruption, the distributions of PCNA and the large subunit of the RFC complex, proteins required for the elongation phase of DNA replicatio n, are altered such that they are found within the intranucleoplasmic lamin aggregates. In contrast, the distribution of XMCM3, XORC2, and D NA polymerase alpha, proteins required for the initiation stage of DNA replication, remains unaltered. The data presented demonstrate that t he nuclear lamins may be required for the elongation phase of DNA repl ication.