Reactivation of DNA Replication in Nuclei from Terminally Differentiated Cells: Nuclear Membrane Permeabilization Is Required for Initiation in XenopusEgg Extract

Abstract

We have used Xenopusegg extract to investigate the requirements for reactivation of DNA replication in nuclei isolated from terminally differentiated chicken erythrocytes. Previous work has shown that reactivation of erythrocyte nuclei in egg extract is accompanied by chromatin decondensation, nuclear envelope reformation, and the accumulation of egg lamin, L III. However, in those studies, erythrocyte nuclei were prepared by methods that were not designed to maintain the selective permeability of the nuclear membrane, and as such, it is not clear if loss of nuclear membrane integrity played a role in the reactivation process. Therefore, the purpose of this study was to determine if changes in nuclear membrane permeability are required for reactivation of erythrocyte nuclei in egg extract. Nuclei with intact nuclear membranes were prepared from erythrocytes with streptolysin O and permeable nuclei by treatment of intact nuclei with the detergent Nonidet-P40. Like permeable nuclei, most intact nuclei decondensed, imported nuclear protein, and accumulated lamin L IIIfrom the extract. However, unlike permeable nuclei, which replicated extensively in the extract, few intact nuclei initiated replication under the same conditions. These data demonstrate that permeabilization of the nuclear membrane is required for reactivation of DNA replication in terminally differentiated erythrocyte nuclei by egg extract and suggest that loss of nuclear membrane integrity may be a general requirement for replication of quiescent cell nuclei by this system.