Replication fork arrest at relocated replication terminators on the Bacillus subtilis chromosome

Abstract

The replication terminus region of the Bacillus subtilis chromosome, comprising TerI and TerII plus the rtp gene (referred to as the terC region) was relocated to serC (257؇) and cym (10؇) on the anticlockwise-and clockwise-replicating segments of the chromosome, respectively. In both cases, it was found that only the orientation of the terC region that placed TerI in opposition to the approaching replication fork was functional in fork arrest. When TerII was opposed to the approaching fork, it was nonfunctional. These findings confirm and extend earlier work which involved relocations to only the clockwise-replicating segment, at metD (100؇) and pyr (139؇). In the present work, it was further shown that in the strain in which TerII was opposed to an approaching fork at metD, overproduction of the replication terminator protein (RTP) enabled TerII to function as an arrest site. Thus, chromosomal TerII is nonfunctional in arrest in vivo because of a limiting level of RTP. Marker frequency analysis showed that TerI at both cym and metD caused only transient arrest of a replication fork. Arrest appeared to be more severe in the latter situation and caused the two forks to meet at ϳ145؇ (just outside or on the edge of the replication fork trap). The minimum pause time effected by TerI at metD was calculated to be ϳ40% of the time taken to complete a round of replication. This significant pause at metD caused the cells to become elongated, indicating that cell division was delayed. Further work is needed to establish the immediate cause of the delay in division.Both Bacillus subtilis and Escherichia coli contain several DNA replication terminators on their chromosomes which, when complexed to their cognate terminator proteins, arrest replication fork movement in a polar manner (4, 7, 9). The terminators are located approximately opposite oriC within a relatively broad region and are organized as two opposed groups. This organization provides a replication fork trap which ensures that the approaching forks always meet within a restricted (terminus) region. The arrangement of terminators on the B. subtilis chromosome is shown in Fig. 1. TerI, III, and V are oriented to block movement of the clockwise fork generated at oriC, while TerII, IV, and VI block the anticlockwise fork (7). TerI and II (originally IRI and IRII) were the first chromosomal terminators to be identified (5). They lie just upstream of the gene (rtp) that encodes the replication terminator protein (RTP). The overall terminus region extends from TerV to TerVI. In the present paper, the chromosomal segment comprising TerI, TerII plus rtp, will be referred to as the terC region because it encompasses the major arrest site, TerI. When this terC region was relocated to the pyr and metD loci (at 139Њ and 100Њ, respectively) it was found that the clockwise fork was arrested at each of these loci only when the orientation of the inserted segment placed TerI in opposition to the approaching clockwise fork (6). TerII was inactive when opposed to the clockwise m...

Termination of DNA Replication in Prokaryotic Chromosomes

“…However, there are some clues as to the possible function of these sequences in cellular metabolism. It is clear that during growth under laboratory conditions, either the six known replication termini or the terminator protein can be deleted without causing loss of cell viability (20,79). On the other hand, the fact that singlepoint mutations can cause loss of function at any terminator site indicates that multiple terminator sites have been preserved by selection pressure against random mutational drift, thus suggesting a selective advantage for cells with a full set of termini.…”

Coupling of σ G Activation to Completion of Engulfment during Sporulation of Bacillus subtilis Survives Large Perturbations to DNA Translocation and Replication

“…5F). In the other, a strain was used in which termination of replication occurred at 145°relative to the origin at 0°on the circular chromosome map, rather than at the normal position, 172° (17,19). Relocating the terminus of replication had no effect on G activation, which occurred only after the completion of engulfment (Table 2; Fig.…”

“…The strain containing the 2.3-Mb insert, SL14378, appeared to be slightly delayed in formation of the sporulation division septum compared to the parent strain. It was considerably delayed in the progression through engulfment stages IIii and IIiii, with half the sporulating population Replication termination occurs at 145°7, 17,19 completing engulfment only about 3 h after they had formed the septum ( Fig. 3B and C).…”

“…We wished to test the effect of disrupting termination of replication, and hence potentially the sequence of translocation events, without large inserts of foreign DNA. For this purpose we used derivatives of a strain (SU227) in which termination of replication occurs at about 145°and not 172°as the result of relocation of key termination loci (17,19). Sporulation efficiency was reduced to about 10%.…”

“…The first approach, delaying replication and translocation, was to use derivatives of B. subtilis with 0.9 and 2.3 Mb of Synechocystis PCC6803 DNA inserted into the 4.2-Mb B. subtilis chromosome (24); the Synechocystis inserts lack rrn genes and appear to be largely silent (24). The second approach, affecting termination of replication and hence potentially the sequence of translocation events, was to use a strain in which a region containing the termination-associated TerI, TerII, and rtp loci had been relocated away from the normal replication termination site at 172°, resulting in termination of replication at 145°relative to the origin of replication at 0° (17,19). The third approach, in which translocation was delayed, was to use a strain with a reduced SpoIIIE DNA translocase activity (3).…”