Functional analysis of the dna (Ts) mutants of Bacillus subtilis: plasmid pUB110 replication as a model system.

We determined the effect of various Bacillus subtilis dna(Ts) mutations on pUB110 and chromosomal replication. Leading strand DNA synthesis of pUB110, starting by a nick at the plasmid replication origin (oriU), is performed by DNA polymerase III, since replication is blocked at non-permissive temperature in thermosensitive mutants dnaD, dnaF, dnaH and dnaN known to cause thermosensitivity of the various subunits of DNA polymerase III. When the lagging strand origin (oriL) is exposed, the DnaG protein (DNA primase) alone, or in association with unknown protein(s) binds asymmetrically to oriL to form a primer that is also extended by DNA polymerase III. In oriL- plasmids like pBT32, leading and lagging strand DNA syntheses are decoupled from each other. The DnaB protein, that is not required for pUB110 replication, may be associated with priming at a second unidentified lagging strand origin on pBT32. At non-permissive temperature, the dnaC30 and dnaI2 mutations affect both pUB110 and chromosomal DNA synthesis.

Functional analysis of the leading strand replication origin of plasmid pUB110 in Bacillus subtilis.

Supercoiled plasmid DNA is the substrate for initiation of pUB110 replication, and - by inference - for binding of its initiator protein (RepU) to the plasmid replication origin (oriU) in vivo. No hairpin structure is required for RepU-oriU recognition. RepH (the pC194 replication initiation protein) failed to initiate replication in trans at oriU. The nucleotides that determine the specificity of the replication initiation process are located within oriU but termination is unefficient. Therefore the segment that forms the full recognition signal for termination is probably located 3' of the oriU recognition sequence. Two overlapping domains, one for initiation and one required for termination, compose the leading strand replication origin of plasmid pUB110.