Breakdown and quantitation of the forked termination of replication intermediate of Bacillus subtilis

Hanley, Peter J.; Carrigan, C.M.; Rowe, Dominic B.; Wake, R.G.

doi:10.1016/0022-2836(87)90043-x

Cited by 17 publications

(8 citation statements)

References 9 publications

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“…It is possible that the cell compensates for the difference in distance by altering the replication rates so that the time required to replicate each arm is roughly the same. A similar effect has been postulated to occur in a B. subtilis strain in which the half-chromosomes differed considerably in length (7).…”

Section: Resultssupporting

confidence: 60%

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

1988

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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...

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Section: Resultssupporting

confidence: 60%

“…Biol. 154: [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][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.…”

mentioning

confidence: 99%

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

1988

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“…The plasmids used have been described previously: pJH101 (4), pLS23-17 (1), pWS8 (21), pWS10 (16), and pWH47 (5). New plasmids were constructed as follows.…”

Section: Methodsmentioning

confidence: 99%

“…That this band represented alforked molecule of the expected dimensions of a band I equivalent, reflecting the arrest of the clockwise fork at terC, was confirmed by testing one of the DNA preparations for its sensitivity to Si nuclease. Si nuclease caused rapid destruction of the putative band I species, with release of one of the expected 15.4-kb arms (5). (In all cases of deletion at the left of terC, where a putative band I species was observed, its sensitivity to Si nuclease was established for at least one of the DNA preparations, but only the data for the more significant SU162 strain are shown; see below.)…”

Section: Methodsmentioning

confidence: 99%

“…The block to movement of the clockwise fork at terC in B. subtilis is very severe, if not complete (5), and the site of the junction between the daughter arms and unreplicated DNA in this arrested fork has been used to define the location of terC. Clearly, the nucleotide sequence in the vicinity of terC, especially but not exclusively that of the unreplicated DNA just ahead of the arrested clockwise fork, must hold at least * Corresponding author.…”

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DNA sequence requirements for replication fork arrest at terC in Bacillus subtilis

Smith

Wake

1988

J Bacteriol

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The replication terminus, terC, of Bacillus subtilis is the chromosomal site at which movement of the clockwise replication fork is blocked. The effect of deletion or modification of DNA sequences on either side of terC (defined by the sequence location of the arrested clockwise fork junction) has been investigated. Deletion of sequences ahead of terC to within 250 base pairs (bp) had no effect on fork arrest, whereas removal of a further 130 bp abolished it. The 250-bp segment immediately ahead of terC encompassed the previously identified inverted repeat region as well as potential promoters for the transcription of an adjoining open reading frame (ORF). Deletion of DNA from the other side of terC up to 80 bp from it also abolished fork arrest. This deletion removed the bulk of the ORF. Disruption of this ORF by the insertion of 4 bp also abolished fork arrest. A model for clockwise fork arrest at terC, implicating both the inverted repeat region and the protein product of the ORF, is proposed.The replication terminus, terC, of the Bacillus subtilis chromosome is located approximately opposite the origin, oriC (23). terC is unique (13,14,18), and at this site movement of the clockwise replication fork, which is the first of the two forks generated at the origin to reach terC, is blocked (8,14,20,21). Clockwise fork arrest appears to represent the first stage in the overall process of termination of replication in B. subtilis. The anticlockwise fork arrives at terC a few minutes later, presumably to fuse with the arrested fork and so complete the process. In Escherichia coli the situation is different. The chromosome contains two regions, Ti and T2, which function as terminators of replication. They are separate by a distance of about 5% of the chromosome and each terminator is polar in its action, causing arrest of just one of the forks, clockwise or anticlockwise (3, 6). Ti has been mapped to within 20 kilobases (kb) and T2 to within 4 kb (7); neither region has been sequenced.terC of B. subtilis has been cloned (16), and recently, a 1.3-kb segment of DNA spanning terC has been sequenced (2). The farthest that a clockwise fork moves within this sequence is to within about 20 nucleotides (at the most) of a region containing two imperfect inverted repeats, each of 47 to 48 nucleotides and separated by 59 nucleotides. (Note that each inverted repeat referred to here could be considered as one-half of a palindrome.) The inverted repeat region is located just upstream of an open reading frame (ORF) which has the potential to code for a protein of 122 amino acids. It is not known whether this ORF is expressed. A diagrammatic representation of these features is shown in the upper portion of Fig. 3 (see below); the clockwise fork enters the region shown from the right.The block to movement of the clockwise fork at terC in B. subtilis is very severe, if not complete (5), and the site of the junction between the daughter arms and unreplicated DNA in this arrested fork has been used to define the location of terC. Clear...

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Replication termini in the rDNA of synchronized pea root cells (Pisum sativum )

et al. 1988

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In synchronized root cells of Pisum sativum (cv. Alaska) the joining of nascent replicons is delayed until cells reach the S–G2 boundary or early G2 phase. To determine if the delayed ligation of nascent chains occurs at specific termination sites, we mapped the location of arrested forks in the ribosomal DNA (rDNA) repeats from cells in late S and G2 phases. Two‐dimensional (neutral‐alkaline) agarose electrophoresis and Southern blot hybridization with specific rDNA sequences show that only cells located at the S–G2 boundary and early G2 phase produce alkali‐released rDNA fragments of discrete size. The released fragments are from a particular restriction fragment, demonstrating that the replication forks stop non‐randomly within the rDNA repeats. Indirect end‐labeling with probes homologous to one or the other end of the fork‐containing restriction fragment shows that there are two termination regions, T1 and T2, where forks stop. T1 is located in the non‐transcribed spacer and T2 is at the junction between the non‐transcribed spacer and the 18S gene. The two termini are separated by ˜1.3 kb. Replication forks stop at identical sites in both the 8.6‐ and 9.0‐kb rDNA repeat size classes indicating that these sites are sequence determined.

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Breakdown and quantitation of the forked termination of replication intermediate of Bacillus subtilis

Cited by 17 publications

References 9 publications

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

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

DNA sequence requirements for replication fork arrest at terC in Bacillus subtilis

Replication termini in the rDNA of synchronized pea root cells (Pisum sativum )

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