REACTIVATION OF DNA-REPLICATION IN NUCLEI FROM TERMINALLY DIFFERENTIATED CELLS - NUCLEAR-MEMBRANE PERMEABILIZATION IS REQUIRED FOR INITIATION IN XENOPUS EGG EXTRACT

We have used Xenopus egg extract to investigate the requirements for r eactivation of DNA replication in nuclei isolated from terminally diff erentiated chicken erythrocytes. Previous work has shown that reactiva tion 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 p repared by methods that were not designed to maintain the selective pe rmeability of the nuclear membrane, and as such, it is not clear if lo ss of nuclear membrane integrity played a role in the reactivation pro cess. Therefore, the purpose of this study was to determine if changes in nuclear membrane permeability are required for reactivation of ery throcyte nuclei in egg extract. Nuclei with intact nuclear membranes w ere prepared from erythrocytes with streptolysin O and permeable nucle i by treatment of intact nuclei with the detergent Nonidet-P40. Like p ermeable nuclei, most intact nuclei decondensed, imported nuclear prot ein, and accumulated lamin L-III from the extract. However, unlike per meable nuclei, which replicated extensively in the extract, few intact nuclei initiated replication under the same conditions. These data de monstrate that permeabilization of the nuclear membrane is required fo r reactivation of DNA replication in terminally differentiated erythro cyte nuclei by egg extract and suggest that loss of nuclear membrane i ntegrity may be a general requirement for replication of quiescent cel l nuclei by this system. (C) 1997 Academic Press.