DNA synthesis in isolated nuclei from synchronized plasmodia of Physarum polycephalum.
Nuclei were isolated from synchronized plasmodia of a true slime mold, Physarum polycephalum, in S-phase, and DNA synthesis in the nuclei was studied in vitro. The nuclei catalyzed DNA synthesis at the rate of 0.7 ng DNA/1.0 X 10(6) nuclei/30 min at 25 degrees C, which was 5 times higher than that catalyzed in G2-phase nuclei. The DNA synthesis required Mg2+, four kinds of deoxyribonucleoside 5'-triphosphates and ATP, suggesting that the mode of synthesis is a replicative-type, but not a repair-one. Sedimentation analysis of the DNA products revealed that the nuclei produced 2-4S DNA fragments mainly during a 30-sec pulse incubation, and 2-4S, 5-12S and longer fragments during a 15-min incubation. The pulse- and chase-labeling experiments showed that the 2-4S fragments shifted discontinuously to longer fragments. These results indicate that the nuclei catalyze the formation of 2-4S Okazaki fragments first and then their subsequent ligation. Eighty % and 96% of the DNA synthesis was inhibited by 200 micrograms/ml aphidicolin and 40 mM N-ethylmaleimide, respectively, but 80% of the activity was resistant to 100 microM 2',3'-dideoxythymidine 5'-triphosphate. These results suggest that the DNA synthesis is catalyzed by the alpha-type DNA polymerase of Physarum polycephalum.
D016590:Aphidicolin; D002453:Cell Cycle; D002467:Cell Nucleus / Q000187:drug effects / Q000648:ultrastructure*; D004261:DNA Replication / Q000187:drug effects; D054306:Dideoxynucleotides; D004224:Diterpenes / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D007399:Interphase; D007700:Kinetics; D010804:Physarum / Q000166:cytology* / Q000254:growth & development; D013942:Thymine Nucleotides / Q000494:pharmacology
D054306:Dideoxynucleotides; D004224:Diterpenes; D013942:Thymine Nucleotides; D016590:Aphidicolin; C017216:2',3'-dideoxythymidine triphosphate; D005033:Ethylmaleimide