The regulation of initiation of chromosome replication in Bacillus subtilis will be investigated . It still is not yet well understood. The mechanism likely involves a multi- component structure inbedded in the cellular membrane which consists of specific DNA fragments and proteins. As for the protein components, the structure and function of the proteins encoded by the dnaB operon genes will be worked out. The function of the dnaB protein is essential for the replication initiation of the chromosome. The gene and its flanking regions have been cloned and sequenced. The dnaB gene turned out to be the first gene of an operon which consists of three or four genes, dnaB, Y-,Z-and possibly genes. Specific antisera against the first three have been prepared and will be used to isolate proteins encoded by the genes of the dnaB operon. Antibodies will be also used for the studies of the in vitro initiation. Attempts will be made to isolate mutants of the Y- and Z-genes. With regard to the DNA fragments involved in the replication initiation, we will study the nature of the two types of membrane binding of the replication origin areas of the B. subtilis chromosome and a plasmid pUB110. The type-I membrane binding is salt-resistant and dependent on the dnaB+ gene function, whereas type-II binding is salt-sensitive and independent of the dnaB+ gene function. It is believed that type I binding is essential for biochemical processes of initiation and type.II binding is important for pUB110 and the chromosomal original to be bound to a structure on the membrane that is essential for both initiation of replication and the partition of daughter chromosomes. The type -I binding site seems to be located at or near the origin of replication both in the B. subtilis chromosome and pUB110. Type-II binding is found in four areas of the pUB110 molecule and in two areas near the purA16 locus that is located within 20.50 kb from the replication origin of the B.subtilis chromosome. The properties and functional significance of these bindings will be analayzed further.
