Replication of nuclei from cycling and quiescent mammalian cells in 6-DMAP-treated Xenopus egg extract.
Nuclear membrane permeabilization is required for replication of quiescent (G0) cell nuclei in Xenopus egg extract. We now demonstrate that establishment of replication competence in G0 nuclei is dependent upon a positive activity present in the soluble egg extract. Our hypothesis is that G0 nuclei lose the license to replicate following growth arrest and that this positive activity is required for relicensing DNA for replication. To determine if G0 nuclei contain licensed DNA, we used the protein kinase inhibitor, 6-dimethylaminopurine (6-DMAP), to prepare egg extracts that are devoid of licensing activity. Intact nuclei, isolated from mammalian cells synchronized in G1-phase (licensed), G2-phase (unlicensed), and G0 were permeabilized and assayed for replication in 6-DMAP-treated and untreated extracts supplemented with [alpha-32P]dATP or biotinylated-dUTP. Very little radioactivity was incorporated 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 similar to the punctate pattern observed within nuclei incubated in aphidicolin-treated extract, suggesting that initiation events occur within most replication factories in 6-DMAP extract. However, density substitution and alkaline gel analyses indicate that the incorporated biotin within these nuclei arises from a small number of active origins which escape 6-DMAP inhibition. We conclude that 6-DMAP-treated egg extract cannot differentiate licensed from unlicensed mammalian somatic cell nuclei and, therefore, cannot be used to determine the "licensed state" of G0 nuclei using the assays described here.
Munshi
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Department of Biochemistry, University of Mississippi Medical Center, Jackson 39216-4505, USA.
Leno
G H
GH
D016475:3T3 Cells; D000225:Adenine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D002453:Cell Cycle; D002457:Cell Extracts; D002467:Cell Nucleus; D002843:Chromatin; D004261:DNA Replication / Q000187:drug effects*; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D008322:Mammals; D051379:Mice; D008807:Mice, Inbred BALB C; D010063:Ovum; D047428:Protein Kinase Inhibitors; D014981:Xenopus
D002457:Cell Extracts; D002843:Chromatin; D004791:Enzyme Inhibitors; D047428:Protein Kinase Inhibitors; C001020:N(6),N(6)-dimethyladenine; D000225:Adenine