Origin-specific DNA-binding membrane-associated protein may be involved in repression of initiation of DNA...

Previous binding studies with labeled double-stranded Bacillus subtilis DNA fragments to a protein blot of renatured Bacillus membrane proteins showed selective binding of two adjacent origin fragments to a 64-kDa protein. The selective binding of the 64-kDa protein could be blocked by prior incubation of the blots with a specific polyclonal antibody. An in vitro replication system derived from a B. subtilis DNA-membrane complex showed initiation activity without addition of exogenous enzymes or template. When the complex was first incubated with the 64-kDa antibody or with its Fab fragments, initiation activity was enhanced. Antibodies to several other Bacillus membrane proteins as well as nonspecific antibodies did not show any significant stimulatory effect. A heavy-density-label experiment indicated that the complex initiated multiple rounds of replication in the presence of the 64-kDa antibody but not in its absence. The 64-kDa antibody plus an initiation inhibitor (streptovaricin) showed only repair and elongation activity. The 64-kDa protein may act in vivo as a repressor/regulator of initiation activity.