DNA synthesis in isolated nuclei from synchronized plasmodia of Physarum polycephalum.

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Abstract
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 .times. 106 nuclei/30 min at 25.degree. 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 .mu.g/ml aphidicolin and 40 mM N-ethylmaleimide, respectively, but 80% of the activity was resistant to 100 .mu.M 2'',3''-dideoxythymidine 5''-triphosphate. These results suggest that the DNA synthesis is catalyzed by the .alpha.-type DNA polymerase of Physarum polycephalum.