Replication arrest

Bacterial chromosomal replication forks are very efficient protein machines, moving along the template and synthesizing DNA at nearly 1000 bp/s. Replication by the two forks assembled at the origin continues at this rate for 40 min to copy the entire 4.7 Mb genome. Therefore, it is not surprising that these replication forks do not simply stop spontaneously when their task is complete. Bacterial Chromosomes Carry Specific Sites and Proteins That Stall Replication Forks In both Escherichia coli and Bacillus subtilis, chromosomal replication initiates from a single origin; two replication forks travel in opposite directions around the circular chromosomes and terminate in a region diametrically opposed from the origin. The terminus regions of both bacteria contain multiple sites that bind specific terminator proteins to cause replication fork arrest (for review see Hill, 1992). The locations of the well-characterized sites (additional sites are still being discovered)on the E. col i and B. subtilis chromosomes are presented in Figure 1. Termination sites stop replication forks only in one orientation. Multiple sites are organized to form a "fork trap" such that replication forks enter but cannot exit the termination zone. For example, in E. coli, the clockwise replication fork passes through TerE, TerD, and TerA, which are in the inactive orientation for this fork, before stopping at TerC, which it meets in the active orientation. Although similar on the surface, the E. coil and B. subtilis termination systems are surprisingly different in detail. The sites responsible for termination in the two bacteria are unrelated, and the proteins that recognize these sites are distinct. E. coil Ter sites are 22 bp and recognized by a monomer of the Tus protein (for terminus utilization substance, also called Tau; molecular weight, 36,000). The analogous sites in B. subtilis are 47 bp"imperfect" inverted repeats (called IR-I and IR-II) that are recognized by the replication terminator protein (RTP). RTP has a monomer molecular weight of 14,500 and forms a stable dimer, and two dimers act together at each termination site. Tus and RTP share little sequence similarity. Most reports state they have no detectable homology, although Bussiere et al. (1995 [this issue of Cel l ] ) suggest a modest relationship in amino acid sequence (22% identity and 40% similarity). The relevance of this sequence similarity has not been investigated. With the exception of a patch of homology between the RTP and the B. subtilis initiation protein DnaB (which is unrelated to E. coli DnaB helicase; see below), the terminator proteins appear unrelated to other known proteins. Below is a summary of the characterization of the two termination systems. This work illuminates how these protein-DNA complexes pause replication forks and the biological consequences of replication termination. E. coli Ter-Tus Complexes The Tus-TerB complex (TeFB is the most extensively studied of the E. coli sites) is very specific (KD, 3.4 X 10 -13 M) and long-lived (half-life of 550 min) (Gottlieb et al., 1992). DNA protection against a variety of reagents indicates that the protein makes asymmetric contacts with the DNA. If the protein-DNA complex is viewed in two halves, the half of the complex proximal to the site of replication fork arrest contains a large number of protected bases on both DNA strands whereas the distal half protects only one strand (Gottlieb et al., 1992; Sista et al., 1991). In vitro, Ter-Tus complexes stall reconstituted replication forks initiated at either the E. coli chromosomal origin (Lee et al., 1989) or the plasmid pBR322 origin (Hill and Marians, 1990). Termination of replication occurs only when forks enter the site in the orientation defined as active in vivo. No proteins other than purified Tus are required, and replication stops at the first nucleotide of the Ter sequence (Hill and Marians, 1990; Lee and Kornberg, 1992). DNA helicases appear to be the replication enzyme targeted for inhibition (Lee et al., 1989; Khatri et al., 1989). Helicases use the energy of ATP hydrolysis to melt the