DNA sequence requirements for replication fork arrest at terC in Bacillus subtilis

Abstract

The replication terminus, terC, of Bacillus subtilis is the chromosomal site at which movement of the clockwise replication fork is blocked. The effect of deletion or modification of DNA sequences on either side of terC (defined by the sequence location of the arrested clockwise fork junction) has been investigated. Deletion of sequences ahead of terC to within 250 base pairs (bp) had no effect on fork arrest, whereas removal of a further 130 bp abolished it. The 250-bp segment immediately ahead of terC encompassed the previously identified inverted repeat region as well as potential promoters for the transcription of an adjoining open reading frame (ORF). Deletion of DNA from the other side of terC up to 80 bp from it also abolished fork arrest. This deletion removed the bulk of the ORF. Disruption of this ORF by the insertion of 4 bp also abolished fork arrest. A model for clockwise fork arrest at terC, implicating both the inverted repeat region and the protein product of the ORF, is proposed.The replication terminus, terC, of the Bacillus subtilis chromosome is located approximately opposite the origin, oriC (23). terC is unique (13,14,18), and at this site movement of the clockwise replication fork, which is the first of the two forks generated at the origin to reach terC, is blocked (8,14,20,21). Clockwise fork arrest appears to represent the first stage in the overall process of termination of replication in B. subtilis. The anticlockwise fork arrives at terC a few minutes later, presumably to fuse with the arrested fork and so complete the process. In Escherichia coli the situation is different. The chromosome contains two regions, Ti and T2, which function as terminators of replication. They are separate by a distance of about 5% of the chromosome and each terminator is polar in its action, causing arrest of just one of the forks, clockwise or anticlockwise (3, 6). Ti has been mapped to within 20 kilobases (kb) and T2 to within 4 kb (7); neither region has been sequenced.terC of B. subtilis has been cloned (16), and recently, a 1.3-kb segment of DNA spanning terC has been sequenced (2). The farthest that a clockwise fork moves within this sequence is to within about 20 nucleotides (at the most) of a region containing two imperfect inverted repeats, each of 47 to 48 nucleotides and separated by 59 nucleotides. (Note that each inverted repeat referred to here could be considered as one-half of a palindrome.) The inverted repeat region is located just upstream of an open reading frame (ORF) which has the potential to code for a protein of 122 amino acids. It is not known whether this ORF is expressed. A diagrammatic representation of these features is shown in the upper portion of Fig. 3 (see below); the clockwise fork enters the region shown from the right.The block to movement of the clockwise fork at terC in B. subtilis is very severe, if not complete (5), and the site of the junction between the daughter arms and unreplicated DNA in this arrested fork has been used to define the location of terC. Clear...

A protein involved in termination of chromosome replication in Bacillus subtilis binds specifically to the terC site

“…There was no evidence for nonspecific interaction of the protein with any fragment when the protein-DNA ratio was twice that needed to completely convert the 209-bp fragment to the slowest derived species (IV) of the ladder (compare lanes 6 and 7). (10). It remains to be established whether or not binding of the RT protein to the IRR per se is sufficient to cause arrest.…”

“…The -200 base pairs (bp) of DNA spanning the IRs is referred to as the IR region (IRR). Experiments with strains in which portions of the sequence on either side of terC were deleted or modified have given information on the sequence requirements for clockwise fork arrest (10). In particular, disruption of the ORF by insertion of four extra nucleotides abolished fork arrest.…”

In vivo characterization of tus gene expression in Escherichia coli

“…Expression would then be repressed once the concentration of Tus had increased sufficiently. A simitar model has been proposed for expression of the replication termination protein of Bacillus subtilis (Smith and Wake, 1988). The cyclic expression of tus could be quite abrupt, due tc the properties of Tus/TerB binding (Gottlieb et al.…”

Crystallization and preliminary X-ray diffraction analysis of theBacillus subtilisreplication termination protein in complex with the 37-base-pair TerI-binding site

“…Replication of the circular chromosome of B. subtilis occurs bidirectionally from oriC, with replication forks migrating until they fuse in a region approximately 180 from the origin. The arrest of replication forks and the coordinated termination of DNA replication is dependent upon the binding of the replication terminator protein (RTP) to its cognate DNA-binding sites known as terminator sites (Ter sites; Smith & Wake, 1988, 1989. Each Ter site comprises two pseudosymmetric binding sites for RTP: a low-affinity A site and a high-affinity B site.…”