Sequence features of the replication terminus of the<i>Bacillus subtilis</i>chromosome

Carrigan, C.M.; Haarsma, J.A.; Smith, Marc D.; Wake, R.G.

doi:10.1093/nar/15.20.8501

Cited by 55 publications

(30 citation statements)

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“…Ter is a conserved 22-bp sequence that inhibits the replication fork in a polar fashion (18). The Bacillus subtilis genome has a counterpart of Ter, called RTS, although there is no similarity between the sequences (5,34). In E. coli, the tus protein binds specifically to the Ter sequence, and the Tus-Ter complex inhibits the action of DNA helicases to arrest replication (14,15,28,32).…”

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The Replication Fork Barrier Site Forms a Unique Structure with Fob1p and Inhibits the Replication Fork

Kobayashi

2003

Molecular and Cellular Biology

158

172

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The replication fork barrier site (RFB) is an ϳ100-bp DNA sequence located near the 3 end of the rRNA genes in the yeast Saccharomyces cerevisiae. The gene FOB1 is required for this RFB activity. FOB1 is also necessary for recombination in the ribosomal DNA (rDNA), including increase and decrease of rDNA repeat copy number, production of extrachromosomal rDNA circles, and possibly homogenization of the repeats. Despite the central role that Foblp plays in both replication fork blocking and rDNA recombination, the molecular mechanism by which Fob1p mediates these activities has not been determined. Here, I show by using chromatin immunoprecipitation, gel shift, footprinting, and atomic force microscopy assays that Fob1p directly binds to the RFB. Fob1p binds to two separated sequences in the RFB. A predicted zinc finger motif in Fob1p was shown to be essential for the RFB binding, replication fork blocking, and rDNA recombination activities. The RFB seems to wrap around Fob1p, and this wrapping structure may be important for function in the rDNA repeats.Sites that cause replication fork pausing have been identified in many genomes and are known as replication fork-blocking sites. The replication fork-blocking site was first identified in bacteria, both in plasmids and in the genome. Plasmid R6K and the genome of Escherichia coli each have a replication termination sequence, Ter (for a review, see reference 18). Ter is a conserved 22-bp sequence that inhibits the replication fork in a polar fashion (18). The Bacillus subtilis genome has a counterpart of Ter, called RTS, although there is no similarity between the sequences (5, 34). In E. coli, the tus protein binds specifically to the Ter sequence, and the Tus-Ter complex inhibits the action of DNA helicases to arrest replication (14,15,28,32). Ter is also known as a recombination hot spot that stimulates both DNA double-strand breaks (37) and recombination (16, 17; for a review, see reference 41). Therefore, one possible role of Ter is thought to be maintenance of genome stability.In eukaryotic cells, replication fork-blocking sites have been identified in the rRNA gene repeats (rDNA) from yeast to human cells (for a review, see reference 41). They are called replication fork barriers (RFB) and inhibit replication forks in the direction opposite to rDNA transcription. The RFB has been studied most intensively in the yeast Saccharomyces cerevisiae. S. cerevisiae carries about 150 copies of the rDNA repeats. The RFB is located near the 3Ј end of the 35S rDNA in one of the nontranscribed spacer (NTS) regions (NTS1) (Fig. 1A). In the other NTS region (NTS2), there is an autonomously replicating sequence (ARS) which functions as a replication origin. In the S phase of the cell cycle, replication starts at the ARS bidirectionally and the rightward-moving replication forks are arrested at the RFB (Fig. 1). However, the other forks can go through the RFB site because of RFB polarity, like for Ter in E. coli (4,29). Therefore, one of the biological roles of the RFB is tho...

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The Replication Fork Barrier Site Forms a Unique Structure with Fob1p and Inhibits the Replication Fork

Kobayashi

2003

Molecular and Cellular Biology

158

172

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“…Originally, two DNA terminators were identified in each of B. subtilis strains 168 and W23 (3,12). Subsequently, four additional terminators in the 168 strain were detected by probing chromosomal DNA with a degenerate oligonucleotide sim-ilar in sequence to the consensus B site of those already identified (5).…”

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Search for additional replication terminators in the Bacillus subtilis 168 chromosome

Griffiths

Wake

1997

J Bacteriol

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The Bacillus subtilis 168 chromosome is known to contain at least six DNA replication terminators in the terminus region of the chromosome. By using a degenerate DNA probe for the consensus terminator sequence and low-stringency hybridization conditions, several additional minor hybridizing bands were identified. DNA corresponding to the most intense of these bands was cloned and characterized. Although localized in the terminus region, it could not bind RTP and possibly represents a degenerate terminator. A search of the SubtiList database identified an additional terminator sequence in the terminus region, near glnA. It was shown to bind RTP and to function in blocking replication fork movement in a polar manner. Its orientation conformed to the replication fork trap arrangement of the other terminators. The low-stringency hybridization experiments failed to identify any terminus region-type terminators in the region of the chromosome where postinitiation control sequences (STer sites) are known to reside. The two most likely terminators in STer site regions, in terms of sequence similarity to terminus region terminators, were identified through sequence searching. They were synthesized and were found not to bind RTP under conditions that allowed binding to terminus region terminators. Neither did they elicit fork arrest, when present in a plasmid, under stringent conditions. It is concluded that the STer site terminators, at least the first two to the left of oriC, do not have the typical consensus A؉B site makeup of terminus region terminators.Escherichia coli and Bacillus subtilis both have a system for arresting a replication fork within the terminus region of their circular chromosomes (6). In each case, fork arrest requires interaction between a specific terminator protein (Tus in E. coli, RTP in B. subtilis) and a short DNA sequence known as a terminator. The terminator protein-DNA terminator complexes are polar in their action (i.e., they arrest a replication fork approaching from one particular direction only). Tus and RTP show no recognizable sequence similarity to each other, and neither do their cognate terminators. Tus functions as a monomer and interacts with a terminator sequence of 22 to 23 bp. The interaction that takes place between RTP and its terminator is more complex. A dimer of RTP binds to each of two adjacent and opposed sites (A and B) which make up a minimal terminator of 29 bp (Fig. 1, top section). This sequence encompasses the segment running from nucleotides (nt) Ϫ2 to 27 of the originally identified terminators (3). The 16-bp A and B sites each comprise a trinucleotide (common to both sites [boxed in Fig. 1]) plus a 13-bp sequence which is different between the two sites. Six terminators (TerI to TerVI) have so far been identified in B. subtilis 168, and two terminators have been identified in B. subtilis W23 (5) (the consensus is shown in Fig. 1). The consensus B site (nt 12 to 27) possesses a detectable level of symmetry about nt 21 (note arrows under G15, A17 and T25, C27 in ...

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“…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).…”

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

Franks

Wake

1996

J Bacteriol

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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...

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Sequence features of the replication terminus of theBacillus subtilischromosome

Cited by 55 publications

References 8 publications

The Replication Fork Barrier Site Forms a Unique Structure with Fob1p and Inhibits the Replication Fork

The Replication Fork Barrier Site Forms a Unique Structure with Fob1p and Inhibits the Replication Fork

Search for additional replication terminators in the Bacillus subtilis 168 chromosome

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

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