The Bacillus subtilis DNA replication terminator.

Abstract

The recent discovery of the Bacillus subtilis plasmid terminator TerLS20 with bidirectional fork arrest activity has provided the opportunity to probe further the structural and functional features of B. subtilis replication terminators in general. The minimal TerI and TerLS20 terminators each comprise two 13 nt segments flanking a central trinucleotide, which is almost completely conserved in all terminators. It corresponds to the region of overlap of the two RTP binding sites (A and B) on the DNA. It has been shown that, despite this conservation, considerable variation in this trinucleotide region still allows fork arrest activity. Thus, the productive interaction of the RTP dimers, which presumably occurs in the vicinity of this trinucleotide region, is not dependent upon stringently defined contacts with the bases in this region. A completely synthetic and highly symmetrical terminator was constructed by replacing the 13 nt segment of the A site of TerI with an opposed segment identical to that in the B site. The efficient bidirectional activity of this new terminator, TerSymB, established more firmly the need for two opposed RTP binding sites in a functional terminator. TerSymB was used to investigate the effect of sequence deviation in one of the 13 nt segments, from that in the B site, on bidirectionality of the terminator. It was found that the deviations introduced converted the terminator significantly towards polarity of action. The partial symmetry within each of the 13 nt segments of TerSymB, and the presumed recognition of this symmetry in the binding of a symmetrical dimer of RTP to each overlapping site, suggest that the bound dimers are centred over positions in the DNA sequence separated by 15 nt. This separation distance has been used in conjunction with the mode of binding of RTP to DNA proposed by Bussiere et al., based on their crystal structure for RTP, to model the interaction of the two dimers of RTP with unbent B-form DNA. Increased separation of the two binding sites of TerSymB was performed by inserting an extra three, seven or ten nucleotides centrally within the TerSymB sequence. The effects of these insertions on RTP binding and fork arrest activity were consistent with the proposed positioning of the RTP dimers within the terminator sequence, and interaction between the dimers bound to TerSymB. A model to account for the generation of RTP-terminator complexes with bidirectional or polar fork arrest activity utilising TerSymB or TerI-VI is presented.