Location of sites that inhibit progression of replication forks in the terminus region of Escherichia coli

Abstract

We used a Southern hybridization assay to locate precisely the sites at which DNA replication is arrested in the terminus region of the Escherichia coli chromosome. The assay was based on the properties of restriction fragments that contain stalled replication forks. Replication forks that entered the terminus from the clockwise direction with respect to the genetic map were inhibited near manA at a site called T2, which we located at kilobase 442 on the physical map of Bouche (J. P. Bouche, J. Mol. Biol. 154:1-20, 1982). Those that entered the terminus region traveling in the counterclockwise direction were inhibited near pyrF at a site called Ti, which we located at kilobase 90. In each case we found only a single, precise site of arrest. Inhibition at Ti was not detectable in our assay in strains lacking the trans-acting locus tus, which is located near T2 and is required for Ti to function. We demonstrated that the sites of inhibition are also used during termination of replication in exponentially growing, wild-type cells. In all previous studies on the terminus of E. coli, inhibition has only been detected in strains that were modified so that the origin used was placed near the terminus to force the use of the sites of inhibition.Termination of DNA replication and correct resolution of the daughter molecules is a critical aspect of the overall process of chromosome replication. This aspect of replication is still poorly understood. One common theme that has emerged is the occurrence of specific sites at which DNA replication is inhibited. There are several examples of such sites in bacterial systems, including the plasmid R6K (3), the chromosome of Bacillus subtilis (16), and the chromosome of Escherichia coli (12,13). In all of these systems, there is a terminus region that is known to inhibit replication, thereby ensuring that termination and resolution occur in that region. In addition to these bacterial systems, inhibition sites have been identified recently in the DNA flanking an intergrated copy of polyomavirus in cultured rat cells (2) and in chromosomal replication in pea plants (7a).In previous experiments it has been demonstrated that in E. coli, replication forks are severely inhibited as they pass through the terminus region between trp (minute 27.7) and manA (minute 35.7), which are approximately opposite the origin of replication (12, 13). A physical and genetic map of this region is presented in Fig. 1 (1, 4).Recently, it has become apparent that termination is more complex than originally thought. The terminus region actually contains two separate regions, referred to as Ti and T2, that inhibit replication forks and that are located at either edge of the terminus region (5, 9). These results were obtained by assays based on DNA-DNA hybridization to determine gene frequencies (5, 9). These assays, to which we refer as marker-frequency assays, were used to monitor replication from inducible origins integrated near the terminus. Replication forks traveling clockwise with respect to the geneti...

Replication Termination inEscherichia coli: Structure and Antihelicase Activity of the Tus-TerComplex

“…The first two termination sites identified were situated at either end of the terminus region (Fig. 1); one was located close to trp at 28 min and the other near manA at 36 min (68,138). This polar block to progress of the fork therefore appeared to be different from that at the R6K terminus, which was known to block fork movement from either direction (11,33,98).…”

“…This problem was solved in 1987 with the realization that the E. coli terminus was made up of discrete loci that separately blocked replication forks moving in opposite directions in a polar manner (38,68,138). The first two termination sites identified were situated at either end of the terminus region (Fig.…”

Termination sites T1 and T2 from the Escherichia coli chromosome inhibit DNA replication in ColE1-derived plasmids

“…DNA was digested with PstI to liberate the cloned fragment, electrophoresed, transferred to nitrocellulose, and probed with the nick-translated 800-bp fragment. If the fragment contained a stalled replication fork, it would migrate more slowly on the gel than the linear fragment would (11,16,17). Such a band was detected only for pAP210 and only with DNA harvested from the tus+ strain ( Fig.…”

“…Ti is located at kb 90 (16 replication from the ColEl origin would not be expected to be inhibited (Fig. 1, pAP211).…”

“…We demonstrated recently that inhibition occurs at precise sites, and we mapped these sites with an accuracy of ±300 base pairs (bp) (16). The site of arrest for counterclockwisetraveling replication forks at Ti is located at kilobase (kb) 90 on the Bouche terminus map (5), which is at min 28 on the genetic map (1).…”