The effect of phospholipase C on DNA synthesis, morphology and phospholipid content of isolated HeLa cell nuclei.

Isolated HeLa cell nuclei have been treated with purified phospholipase C (Bacillus cereus) and sphingomyelinase (Staphylococcus aureus). The phospholipids of untreated nuclei consisted of about 67% phosphatidylcholine, 23% phosphatidylethanolamine, 7% sphingomyelin, 2% phosphatidylserine and 1% phosphatidylinositol. Phospholipase C degraded 80-90% of the total phospholipids of the nuclei. Such nuclei seemed ultrastructurally intact, and had an average diameter and a protein loss during incubation which were not significantly different from those of controls. Their rate of DNA synthesis was only slightly reduced after treatment with phospholipase C alone and slightly more reduced when phospholipase C was used in combination with sphingomyelinase. This suggests that the polar head-groups of the nuclear phospholipids are of very limited importance in DNA synthesis. Since it has been reported that phospholipase C treatment releases nascent DNA from a membrane complex, the absence of a concommitant reduction in DNA synthesis may suggest that this complex is not necessary for the replication of DNA. Phospholipase C did not significantly influence the stability of the DNA product and gave only a slight inhibition of cytosol and nuclear DNA polymerases when tested with exogenous template.