The terminus region of the Escherichia coli chromosome contains two separate loci that exhibit polar inhibition of replication.

Abstract

The terminus region of the chromosome of Escherichia coli contains two separate sites, called Ti and T2, that inhibit replication forks. Ti is located near 28.5 min, which is adjacent to tip, and T2 is located at 34.5-35.7 min on the opposite side of the terminus region, near manA. The sites act in a polar fashion, and replication forks traveling in a clockwise direction with respect to the genetic map are not inhibited as they pass through Ti but are inhibited at T2. Similarly, counterclockwise forks are not inhibited at T2 but are inhibited at Ti. Consequently, forks are not inhibited until they have passed through the terminus region and are about to leave it. Studies with deletion strains have located T2 within a 58-kilobase interval, which corresponds to kilobase coordinates 387-445 on the physical map of the terminus region.The terminus region of the chromosome of Escherichia coli is located directly opposite the origin of replication on the circular genetic map (1). One of the most interesting features of this region is that it inhibits replication forks that are traveling in either a clockwise or counterclockwise direction with respect to the map (2-5). When origins of replication located near the terminus region were used (2-5), inhibition of replication forks occurred somewhere in the interval between trp (28 min) and manA (36 min). Although the function of this inhibition is unknown, its presence suggests that the terminus might encode partitioning, decatenation, or cell-division control sites, and impediment of replication forks in this region evolved to ensure efficient use of such sites (6).More recent experiments using replication cycles initiated at oriC (7) indicated that the last DNA to be replicated, and consequently the region where forks meet most frequently, was located near 31.2 min. Based on these results, it generally has been assumed that the replication block (terC) is located at this position (1). As discussed by Bouchd et al. (7), however, other interpretations of the data can be made. Therefore, identification of the region of most frequent fork encounter does not necessarily identify the site ofreplicationfork inhibition.We report here, as do de Massy et al. in an accompanying report in this issue (8), that replication forks are not inhibited at a single site in the middle of the terminus region. Instead, there are two inhibition sites, Ti and T2, which are located at the outer edges of the terminus region. These sites are polar, and they only inhibit replication forks that have passed through the terminus region and are about to leave it. Specifically, clockwise traveling replication forks are inhibited at T2 at 34.5-35.8 min, near manA, and counterclockwise traveling forks are inhibited at Ti at 28.5 min, near trp. MATERIALS AND METHODSBacterial Strains. All replication fork assays were performed with strains PK998 or PK1012 and derivatives containing the indicated deletions. These temperature-sensitive strains are dnaA mutants and contained temperature-sensitive P2 D4 cS...

The Escherichia coli Tus–Ter replication fork barrier causes site-specific DNA replication perturbation in yeast

“…Tus binds asymmetrically to Ter and presents either a 'restrictive' (blocking) or 'permissive' (non-blocking) face to the advancing replisome. Ter sites are arranged in the E. coli genome such that Tus binding to Ter creates a polar RF barrier that acts to 'trap' the two converging RFs in a defined DNA replication termination zone that is diametrically opposite to the DNA replication origin (oriC) in the E. coli genome 17 .…”

Strand separation establishes a sustained lock at the Tus–Ter replication fork barrier

“…The forks progress at an average speed of 1 kbp/s until they meet again at the terminus region. As the replication forks approach the terminus, each encounters five 23 bp Ter DNA sites bound in a specific orientation by a 36 kDa DNA binding protein called Tus [1][2][3][4] , and proceeds unhindered. However, when a replication fork continues beyond the terminus, Tus-Ter is approached from the opposite direction (Fig.…”

“…However, when a replication fork continues beyond the terminus, Tus-Ter is approached from the opposite direction (Fig. 1a), triggering Tus-Ter to form a tightly locked complex, thereby bringing the replication fork to a halt 1,[5][6][7] . Each Ter site is non-palindromic, does not contain any direct repeats and has a strictly conserved GC6 base pair followed by a highly conserved 13 base-pair core region.…”

The Escherichia coli terB sequence affects maintenance of a plasmid with the M13 phage replication origin

“…These sequences impede replication initiated at the E. coli chromosomal origin or at the replication origins of phages or plasmids integrated into the chromosome (4,5,10). Homologous terminator sites were also identified on E. coli plasmid R6K (1,15).…”

“…The replication terminus of the Escherichia coli chromosome is a large region (350 kb) flanked on both sides by terminator sites that arrest replication in a polar fashion (4,10). These sequences impede replication initiated at the E. coli chromosomal origin or at the replication origins of phages or plasmids integrated into the chromosome (4,5,10).…”