INITIATION OF CHROMOSOME-REPLICATION - STRUCTURE AND FUNCTION OF ORIC AND DNAA PROTEIN IN EUBACTERIA

Recent advances in DNA technology have made it possible to analyse the structure and function of the replication origin region of the chromosomes of various bacteria. Comparative studies have shown that 2 basic elements, the replicator and initiator, involved in initiation of chromosome replication are common to most eubacteria but with differences in the fine organization of these elements. In this article, we first review studies of the structural analysis of the origin regions of bacterial chromosomes, and then we summarize our recent work on the function of the 2 elements in Bacillus subtilis as compared to Escherichia coli, in order to show how organization of the elements is related to the differences in regulation of the initiation of replication in the 2 bacteria. Remarkable conservation of genes and their organization in the replication origin region was found in 5 bacteria representative of 3 major branches of the bacterial phylogenic tree. It was concluded that the conserved region containing the dnaA gene is the replication origin of the ancestral bacterium. Conservation of DnaA protein and its binding sequence (DnaA box) is remarkable, suggesting that they are the initiator and replicator of the chromosomes of most eubacteria. We have recently isolated an autonomously replicating sequence (ars) from B. subtilis. The essential features of ars, the presence of DnaA boxes and repeats of an AT rich 15-mer, are the same as E. coli oriC. However, 2 DnaA-box regions flanking the dnaA gene are both required for B. subtilis ars. The function of DnaA protein in vivo was studied in detail using a temperature-sensitive dnaA mutant in B. subtilis. It was clearly shown that DnaA protein is not only essential for initiating chromosome replication but is also a limiting factor for determining the frequency of initiation in B. subtilis. Striking differences in the regulation of initiation are found between B. subtilis and E. coli. DnaA-box regions are incompatible in B. subtilis, but not in E. coli. In consequence, oriC plasmid can exist in only one copy in the former but in multiple copies in the latter. Overexpression of DnaA stimulates over-initiation in E. coli, but inhibits initiation in B. subtilis. The molecular basis for these differences are discussed in terms of differences in number and organization of DnaA boxes and the dnaA gene in the 2 bacteria.