INITIATION OF DNA-REPLICATION IN BACILLUS-SUBTILIS .5. ROLE OF DNA GYRASE AND SUPERHELICAL STRUCTURE IN INITIATION

When spores of a thymine-requiring mutant of B. subtilis were germinated in a medium lacking thymine, an initiation potential (an ability to initiate and complete 1 round of replication in the presence of thymine and in the absence of protein and RNA synthesis) was formed for both chromosomal and plasmid replication. The effect of 2 inhibitors of DNA gyrase, novobiocin (Nov) and nalidixic acid (Nal), on the initiation potential formed during germination for chromosomal and plasmid replication was examined. Nov and Nal inhibited formation of the initiation potential completely if the drug was added at the onset of germination. Initiation of chromosomal and plasmid replication occurred in the presence of DNA gyrase inhibitors when the drug was added after the initiation potential had been fully formed. Chromosomal replication initiated in the presence of the inhibitors ceased after a fragment of .apprx. 15 Md [megadaltons] had been replicated, and plasmid replication was limited to 1 round of replication in approximately half of the plasmid molecules present in the spores. The initiation potential for both chromosomal and plasmid replication, though established, was destroyed gradually but steadily by prolonged incubation with Nov in the absence of thymine. Relaxation of the superhelical structure of plasmid DNA during incubation with Nov was observed in vivo. This relaxation was blocked by ethidium bromide, which dissociated the S-complex. Incubation with Nal did not reduce the initiation potential nor did it change the superhelicity of the plasmid DNA in vivo. This is consistent with the known effect of gyrase inhibitors on the enzymatic activity of DNA gyrase. The action of DNA gyrase and the superhelical structure of the DNA are apparently essential for the initiation of chromosomal and plasmid replication. The specific chromosome organization essential for initiation and elongation and the role of DNA gyrase are discussed.