ORIGIN OF REPLICATION OF THE BACILLUS-SUBTILIS CHROMOSOME - IN-VITRO APPROACH TO THE ISOLATION OF EARLY REPLICATING SEGMENTS

We have developed a permeable cell system for the study of the molecul ar mechanisms involved in the control and initiation of DNA replicatio n at the origin of the Bacillus subtilis chromosome, Our system takes advantage of the synchronous initiation of DNA replication that occurs in outgrowing, subtilis spores and the curtailment of DNA elongation by novobiocin. Early replicating DNA sequences were identified by the use of 5-mercury-dCTP as substrate, which allows the isolation of nasc ent DNA chains by affinity chromatography on thiol agarose. The averag e size of the isolated nascent DNA was 1,000 bp, and more than 80% of the nascent DNA chains had RNA primers at their 5' end, The study of t he temporal order of chromosome replication near the origin using this experimental system showed that a segment containing recF and gyrB re plicated earlier than a segment containing gyrA and part of the rRNA o peron (rrnO). This observation is in agreement with previous in vivo d ata on the replication of origin region and supports the conclusion th at the major activity in our in vitro system was the faithful replicat ion of the ori region.