Biochemical Study on the Initiation Complex of the Replication of the Bacillus Subtilisin Chromosome

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1. Formation of a unique protein-DNA complex (initiosome) by a sequential action of DnaA gene products and oriC is thought to be prerequisite for initiation of chromosomal replication in bacteria. We previously found that DnaA protein seems to play a central role in initiation of replication in bacterial species diverged widely in evolution. However, regulatory mechanism which determines the time of activation of oriC by DnaA protein wa not known. The aim of this research was to unveil the organization of initiosome in order to understand the regulatory mechanism of initiation of replication.2. We previously found that combination of the DnaA gene and its binding sequence (DnaA-box) is conserved widely in eubacteria, and that Bacillus subtilisin DnaA protein binds specifically to DnaA-box in vitro. We now isolated a temperature sensitive mutant of the B. subtilisin DnaA, and showed directly that DnaA protein is specifically involved in initiation of chromosomal replication. In addition … More , we demonstrated that a DNA fragment containing two DnaA-box clusters flanking DnaA gene has ars activity in B. subtilisin.3. The mutant DnaA protein was unstable at the unpermissive temperature. Its amount decreased rapidly during the first incubation period at 49 C, and then started to increase at the elevated level probably due to the derepression of autogenous regulation. This phenomena indicated that inactivation of DnaA protein causes the destruction of the initiosome, and then increasing its sensitivity to protease action. Taking advantage of this phenomena, we showed that there is a linear relationship between the amount of DnaA protein and cell's initiation potential.4. On the other hand, we found that the specific inhibition of initiation of chromosomal replication occurs by the artificial overproduction of the DnaA protein in B. subtilisin. This result indicates that there are other limiting factors for initiation of replication, which will work in concert with the DnaA protein. In order to elucidate such factors, biochemical analysis of proteins which forms a complex with the DnaA protein and genetic analysis of suppressor mutations of the DnaA mutation are now in progress. Less