REPLICATION OF NUCLEI FROM CYCLING AND QUIESCENT MAMMALIAN-CELLS IN 6-DMAP-TREATED XENOPUS EGG EXTRACT

Nuclear membrane permeabilization is required for replication of quies cent (GO) cell nuclei in Xenopus egg extract. We now demonstrate that establishment of replication competence in GO nuclei is dependent upon a positive activity present in the soluble egg extract. Our hypothesi s is that GO nuclei lose the license to replicate following growth arr est and that this positive activity is required for relicensing DNA fo r replication. To determine if G0 nuclei contain licensed DNA, we used the protein kinase inhibitor, 6-dimethylaminopurine (6-DMAP), to prep are egg extracts that are devoid of licensing activity. Intact nuclei, isolated from mammalian cells synchronized in G1-phase (licensed), Ga -phase (unlicensed), and GO were permeabilized and assayed for replica tion in 6-DMAP-treated and untreated extracts supplemented with [alpha -P-32]dATP or biotinylated-dUTP. Very little radioactivity was incorpo rated into nascent DNA in each nuclear population; however, nearly all nuclei in each population incorporated biotin in 6-DMAP extract. The pattern of biotin incorporation within these nuclei was strikingly sim ilar to the punctate pattern observed within nuclei incubated in aphid icolin-treated extract, suggesting that initiation events occur within most replication factories in 6-DMAP extract. However, density substi tution and alkaline gel analyses indicate that the incorporated biotin within these nuclei arises from a small number of active origins whic h escape 6-DMAP inhibition, We conclude that 6-DMAP-treated egg extrac t cannot differentiate licensed from unlicensed mammalian somatic cell nuclei and, therefore, cannot be used to determine the ''licensed sta te'' of GO nuclei using the assays described here. (C) 1998 Academic P ress.