INITIATION OF DNA-REPLICATION IN BACILLUS-SUBTILIS .3. ANALYSIS OF MOLECULAR EVENTS INVOLVED IN INITIATION USING A TEMPERATURE-SENSITIVE DNA MUTANT

[摘要] A temperature-sensitive mutant of B. subtilis, CRK2001, defective in the initiation of DNA replication is described. When the temperature was raised, initiation of DNA replication was immediately prevented, while on-going replication continued to complete rounds of replication. Reinitiation occurred from the normal replication origin 10-15 min after cells heated to 47.degree. C were cooled to 30.degree. C. The reinitiation did not require protein synthesis but did require de novo RNA synthesis. The ability of the cell to reinitiate replication at 47.degree. C or in the presence of rifampicin returned rapidly at 30.degree. C. Using a rifampicin-reversible derivative of CRK2001, a temperature-resistant initiation potential (cell''s ability for initiating replication at 47.degree. C) was formed at 30.degree. C just as rapidly in the presence of rifampicin as in its absence. This indicates that reactivation of a temperature-sensitive product (Pts27) and de novo synthesis of initiation RNA are required for the reinitiation. I[initiation]-RNA synthesis occurred at 47.degree. C and at 30.degree. C, but it turns over rapidly with a half-life of 2 min in the absence of the active gene product. The half-life of I-RNA in this state was approximately the same as that of average mRNA. A completed initiation potential was formed at 30.degree. C in the absence of thymine. It remained stable at 47.degree. C but decayed slowly in the presence of rifampicin. The half-life of RNA in this stage was 12 min and was much longer than that of I-RNA synthesized in the absence of active gene product. A sequence of events involved in the formation of the initiation potential in this mutant was constructed based on these observations.