Sequence limits of DNA strands in the arrest replication fork at the Bacillus subtilis chromosome terminus.

The DNA sequence limits of the leading and lagging strands in the arrested clockwise replication fork at the terminus of the Bacillus subtilis chromosome have been investigated. On the basis of hybridization to synthetic oligonucleotides corresponding to known positions in the terminus region sequence it has been shown that neither the leading nor lagging strands, as they approach terC, traverse the distal inverted repeat, IRI. But a small fraction of the leading strands pass through the proximal inverted repeat, IRII. This is consistent with IRI being the functional inverted repeat in arresting the clockwise fork. But most of the forks appear to stop at least 100 nucleotides short of IRI, and at various positions extending over a distance of at least 100 nucleotides.     

DNA and protein sequence conservation at the replication terminus in Bacillus subtilis 168 and W23.

Cloned DNA from the replication terminus region of Bacillus subtilis 168 was used to identify and construct a restriction map of the homologous region in B. subtilis W23. With this information, DNA from the terminus region of W23 was cloned and the sequence was determined for a 1,499-base-pair segment spanning the expected terC site. The position of the site was then located more precisely. Use of the cloned DNA from strain W23 as a probe for digests of DNA from exponentially growing cells of the same strain established the presence of the slowly migrating replication termination intermediate (forked DNA). The orientation and dimensions of the forked molecule were consistent with arrest of the clockwise fork at the terC site in W23, as has been shown to occur in strain 168. Thus, despite significant differences between the two strains, the same termination mechanism appears to be used. The DNA sequences spanning the terC site in strains 168 and W23 showed a high level of homology (90.2%) close to the site but very little at a distance of approximately 250 base pairs from the site in one particular direction. The overall sequence comparison emphasised the importance of the open reading frame for a 122-amino-acid protein adjacent to terC. Although there were 22 base differences in the open reading frames between the strains, the amino acid sequence of the encoded protein was completely conserved. It is suggested that the amino acid sequence conservation reflects a role for the protein in the clockwise fork arrest mechanism as proposed earlier (M.T. Smith and R.G. Wake, J. Bacteriol. 170:4083-4090, 1988).     

Relocation of the replication terminus, terC, of Bacillus subtilis to a new chromosomal site

The terC-deletion strain of Bacillus subtilis 168, SU153 [Iismaa and Wake, J. Mol. Biol. 195 (1987) 299-310] was used for the re-insertion of a 1.75-kb segment of DNA containing terC at a site approx. 25 kb from its original position. The relocated terC in the new strain, SU160, was oriented normally with respect to the approaching clockwise replication fork, and was positioned such that this fork was the first to reach it. The relocated terC was effective in causing arrest of the clockwise fork, as evidenced by the appearance of a unique DNA species with a characteristic mobility in agarose gel electrophoresis and with a predicted single-strand composition. Thus, the previously cloned 1.75-kb terC-containing segment [Smith et al., Gene 38 (1985) 9-17] has not been altered with respect to TerC function and contains sufficient sequence for this function. The findings reported here provide the opportunity for establishing the minimal and essential sequence features of terC, and for examining its possible polarity of action in causing fork arrest.     

Expression of the rtp gene of Bacillus subtilis is required for replication fork arrest at the chromosome terminus

It was earlier proposed that clockwise replication fork arrest at the chromosome terminus in Bacillus subtilis is dependent upon expression of the rtp gene adjacent to the site of arrest, terC [Smith and Wake, J. Bacteriol. 170 (1988) 4083-4090]. A merodiploid strain of B. subtilis, in which rtp was placed under the control of the IPTG-inducible spac-1 promoter, was constructed. Replication fork arrest at terC, as monitored by the level of a forked DNA molecule of predicted dimensions, was shown to be dependent upon IPTG-induced expression of rtp in this strain. The very low concentration of IPTG needed to induce a substantial level of fork arrest suggests that relatively little RTP, the protein product of rtp, is needed for fork arrest at terC.     

The oriT region of the conjugative transfer system of plasmid pCU1 and specificity between it and the mob region of other N tra plasmids.

The oriT region of the conjugative IncN plasmid pCU1 has been localized to a 669-bp sequence extending from pCU1 coordinates 8.48 to 9.15 kb. The nucleotide sequence of this region was determined. The region is AT-rich (69% AT residues), with one 19-bp and one 81-bp sequence containing 79% or more AT residues. Prominent sequence features include one set of thirteen 11-bp direct repeats, a second set of two 14-bp direct repeats, six different inverted repeat sequences ranging from 6 to 10 bp in size, and two sequences showing 12 of 13 nucleotides identical to the consensus integration host factor binding sequence. Specificity between this oriT and mobilization (mob) functions encoded by the N tra system was demonstrated. This specificity is encoded by the region lying clockwise of the BglII site at coordinate 3.3 on the pCU1 map. Two N tra plasmids isolated in the preantibiotic era were unable to mobilize recombinant plasmids carrying the oriT region of pCU1 or to complement transposon Tn5 mutations in the mob region of the closely related plasmid pKM101.     

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

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 degrees) and cym (10 degrees) 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 degrees) and pyr (139 degrees). 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 approximately 145 degrees (just outside or on the edge of the replication fork trap). The minimum pause time erected by TerI at metD was calculated to be approximately 40% 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.     

Definition and polarity of action of DNA replication terminators in Bacillus subtilis.

The first stage in termination of chromosome replication in Bacillus subtilis involves arrest of the clockwise fork at the inverted repeat region (IRR), comprising the opposed IRI and IRII sequences, adjacent to the upstream region of the rtp gene, which encodes the replication terminator protein RTP. RTP binds to IRI and IRII. The ability of the IRR and its components to function as terminators, in conjunction with RTP, and their polarity of action have now been tested by the use of plasmids replicating in B. subtilis as unidirectional theta structures and into which potential terminator sequences were inserted in alternate orientations relative to fork movement. When the complete IRR was inserted into such plasmids and the new plasmids transferred into a B. subtilis strain overproducing RTP, it was able to block movement of a replication fork approaching from either direction. IRI and IRII were shown to function as polar terminators, each blocking movement of a fork when it approached from one particular direction but not the other. Furthermore, the polarity of action was in accordance with the IRR being able to operate as a replication fork trap. Thus, a fork approaching the IRR would pass through the first terminator encountered (IRI or IRII) and be halted by the second. The previously observed nonfunctioning of a particular orientation of chromosomal IRR as a fork arrest site probably reflects a limiting level of RTP in the cell. Interestingly, a 21 base-pair core sequence spanning a single RTP binding site within IRI (the 47 base-pair IRI contains 2 binding sites) was unable to arrest a fork approaching from either direction in the plasmid system. This suggests that both binding sites within an IR must be filled in order to function as an arrest site. It is possible that co-operative interaction between adjacent dimers within IRI or IRII provides the necessary conformation for causing fork arrest.     

Identification of the replication terminator protein binding sites in the terminus region of the Bacillus subtilis chromosome and stoichiometry of the binding

DNase I footprinting of the interaction between the replication terminator protein (RTP) of Bacillus subtilis and the inverted repeat region (IRR) at the chromosome terminus, to which it binds to block the clockwise replication fork, showed that two major regions of 41 base pairs (bp) were protected from cleavage. These regions corresponded approximately to the imperfect inverted repeats (IRI and IRII) identified previously. Band retardation analyses of the interaction between RTP and portions of the IRR established that each inverted repeat (IRI or IRII) contained two RTP binding sites. By sedimentation equilibrium in the ultracentrifuge, RTP was found to exist as a dimer of 29 kDa at neutral pH and concentrations above 0.2 g/l. Quantitative studies of the RTP-IRR interaction using [3H]RTP and [32P]IRR showed that the fully saturated complex contained eight RTP monomers per IRR. It is concluded that a dimer of RTP binds to each of the four sites in IRR. The apparent dissociation constant for the interaction was estimated (in the presence of 50% glycerol) to be 1.2 x 10(-11) M (dimer of RTP). Glycerol was found to have a marked effect on the affinity of RTP for the IRR and on the relative amounts of the interaction complexes formed; in the absence of glycerol the dissociation constant was approximately 50-fold higher and there was pronounced co-operative binding of RTP dimers to adjacent sites in each inverted repeat. Examination of the DNA sequence in IRI and IRII identified two 8 bp direct repeats in each. The regions protected from DNase I cleavage in each inverted repeat and the protection afforded by a core sequence spanning just one of the 8 bp direct repeats were consistent with each 8 bp repeat representing a recognition sequence for the RTP dimer. A model describing the binding of RTP to the IRR is presented.     

Normal terC-region of the Bacillus subtilis chromosome acts in a polar manner to arrest the clockwise replication fork

A procedure is described for relocating a functional terC-region to various sites on the Bacillus subtilis chromosome, and in alternative orientations. The relocated terC-region comprised the IRR-rtp portion of the chromosome contained within a 1.75 x 10(3) base-pair segment of DNA. This segment was first cloned into the Tn 917 vector pTV20 in both orientations, and the two new plasmids used for inserting the terC-region into chromosomal copies of Tn 917. When relocated to the pyr and metD loci (139 degrees and 100 degrees positions on the 360 degrees map) it was found that clockwise replication fork arrest occurred only when the IRR-rtp (or terC-) region was oriented, in relation to the direction of approach of the fork, in the same way as in the wild-type strain. Thus, the complete IRR when located in the chromosome, and apparently made up of opposing terminators which might enable it to function in both orientations, is polar in its action. Of the two inverted repeats present in the IRR, it appears that IRI is functional in the chromosome, but not IRII.     

Analysis of insertion into secondary attachment sites by phage lambda and by int mutants with altered recombination specificity

When phage lambda lysogenizes a cell that lacks the primary bacterial attachment site, integrase catalyzes insertion of the phage chromosome into one of many secondary sites. Here, we characterize the secondary sites that are preferred by wild-type lambda and by lambda int mutants with altered insertion specificity. The sequences of these secondary sites resembled that of the primary site: they contained two imperfect inverted repeats flanking a short spacer. The imperfect inverted repeats of the primary site bind integrase, while the 7 bp spacer, or overlap region, swaps strands with a complementary sequence in the phage attachment site during recombination. We found substantial sequence conservation in the imperfect inverted repeats of secondary sites, and nearly perfect conservation in the leftmost three bases of the overlap region. By contrast, the rightmost bases of the overlap region were much more variable. A phage with an altered overlap region preferred to insert into secondary sites with the corresponding bases. We suggest that this difference between the left and right segments is a result of the defined order of strand exchanges during integrase-promoted recombination. This suggestion accounts for the unexpected segregation pattern of the overlap region observed after insertion into several secondary sites. Some of the altered specificity int mutants differed from wild-type in secondary site preference, but we were unable to identify simple sequence motifs that account for these differences. We propose that insertion into secondary sites is a step in the evolutionary change of phage insertion specificity and present a model of how this might occur.     

Insertion and deletion mutations in the repA4 region of the IncFII plasmid NR1 cause unstable inheritance.

Mutants of IncFII plasmid NR1 that have transposons inserted in the repA4 open reading frame (ORF) are not inherited stably. The repA4 ORF is located immediately downstream from the replication origin (ori). The repA4 coding region contains inverted-repeat sequences that are homologous to the terC inverted repeats located in the replication terminus of the Escherichia coli chromosome. The site of initiation of leading-strand synthesis for replication of NR1 is also located in repA4 near its 3' end. Transposon insertions between ori and the right-hand terC repeat resulted in plasmid instability, whereas transposon insertions farther downstream did not. Derivatives that contained a 35-bp frameshift insertion in the repA4 ORF were all stable, even when the frameshift was located very near the 5' end of the coding region. This finding indicates that repA4 does not specify a protein product that is essential for plasmid stability. Examination of mutants having a nest of deletions with endpoints in or near repA4 indicated that the 3' end of the repA4 coding region and the site of leading-strand initiation could be deleted without appreciable effect on plasmid stability. Deletion of the pemI and pemK genes, located farther downstream from repA4 and reported to affect plasmid stability, also had no detectable effect. In contrast, mutants from which the right-hand terC repeat, or both right- and left-hand repeats, had been deleted were unstable. None of the insertion or deletion mutations in or near repA4 affected plasmid copy number. Alteration of the terC repeats by site-directed mutagenesis had little effect on plasmid stability. Plasmid stability was not affected by a fus mutation known to inactivate the termination function. Therefore, it appears that the overall integrity of the repA4 region is more important for stable maintenance of plasmid NR1 than are any of the individual known features found in this region.     

A complex repeated DNA sequence within the Drosophila transposable element copia.

A 320 nucleotide repeated DNA sequence within the copia coding element of Drosophila melanogaster has been identified and characterized. This sequence has been localized by DNA-DNA hybridization and electron microscopic analysis of heteroduplexes to the approximate middle of the 5 kb copia coding region. The primary sequence of this repeated DNA has been determined. The sequence is composed of three related subunits, 35-37 nucleotides in length (A, B and C). This 105 nucleotide higher order repeat has apparently been duplicated twice to yield a complex repeated sequence, ABCA'B'C'A"B"C", which exhibits divergence among the individual subunits. This sequence is AT rich, as are the direct terminal repeats which flank the copia coding region, but does not contain any apparent homology with the terminal repeats. This repeated sequence contains three presumptive polyadenylation signals and two 25 nucleotide, imperfectly matched, inverted repeat sequences adjacent to two of the polyadenylation sequences.     

Pogo transposase contains a putative helix-turn-helix DNA binding domain that recognises a 12 bp sequence within the terminal inverted repeats.

Pogo is a transposable element with short terminal inverted repeats. It contains two open reading frames that are joined by splicing and code for the putative pogo transposase, the sequence of which indicates that it is related to the transposases of members of the Tc1/mariner family as well as proteins that have no known transposase activity including the centromere binding protein CENP-B. We have shown that the N-terminal region of pogo transposase binds in a sequence-specific manner to the ends of pogo and have identified residues essential for this. The results are consistent with a prediction that DNA binding is due to a helix-turn-helix motif within this region. The transposase recognises a 12 bp sequence, two copies of which are present at each end of pogo DNA. The outer two copies occur as inverted repeats 14 nucleotides from each end of the element, and contain a single base mismatch and indicate the inverted repeats of pogo are 26 nucleotides long. The inner copies occur as direct repeats, also with a single mismatch.     

Nucleotide sequence analysis of the long terminal repeat of integrated bovine leukemia provirus DNA and of adjacent viral and host sequences.

The nucleotide sequence of the 3' long terminal repeat and adjacent viral and host sequences was determined for a bovine leukemia provirus cloned from a bovine tumor. The long terminal repeat was found to comprise 535 nucleotides and to harbor at both ends an imperfect inverted repeat of 7 bases. Promoter-like sequences (Hogness box and CAT box), an mRNA capping site, and a core enhancer-related sequence were tentatively located. No kinship was detected between this bovine leukemia proviral fragment and other retroviral long terminal repeats, including that of human T-cell leukemia virus.     

Three transposed elements in the intron of a human VK immunoglobulin gene.

Two gene segments coding for the variable region of human immunoglobulin light chains of the kappa type (VK genes, ref. 2) were found to have unusual structures. The two genes which are called A6 and A22 are located in duplicated gene clusters. Their restriction maps are very similar. About 4 kb of the A22 gene region were sequenced. It turned out that the intron contains an insert with the characteristics of a transposed element. The inserted DNA of 1.2 kb length contains imperfect direct and inverted repeats at its ends; at the insertion site a duplication of five nucleotides was found. Within the inserted DNA one copy each of an Alu element and of the simple sequence motif (T-G)17 were identified. Also these two repetitive sequences are themselves flanked by short direct repeats. The major inserted DNA has no significant homology to published human nucleic acid sequences. The whole structure is interpreted best by assuming a sequential insertion of the three elements. The coding region of the VK gene itself has several mutations which by themselves would render it a pseudogene; we assume that the insertion event(s) occurred prior to the mutations. According to mapping and hybridization data A6 is very similar to A22.     

Structure of the gene encoding the exoglucanase of Cellulomonas fimi

In Cellulomonas fimi the cex gene encodes an exoglucanase (Exg) involved in the degradation of cellulose. The gene now has been sequenced as part of a 2.58-kb fragment of C. fimi DNA. The cex coding region of 1452 bp (484 codons) was identified by comparison of the DNA sequence to the N-terminal amino acid (aa) sequence of the Exg purified from C. fimi. The Exg sequence is preceded by a putative signal peptide of 41 aa, a translational initiation codon, and a sequence resembling a ribosome-binding site five nucleotides (nt) before the initiation codon. The nt sequence immediately following the translational stop codon contains four inverted repeats, two of which overlap, and which can be arranged in stable secondary structures. The codon usage in C. fimi appears to be quite different from that of Escherichia coli. A dramatic (98.5%) bias occurs for G or C in the third position for the 35 codons utilized in the cex gene.     

Sequence of 1019 nucleotides encompassing one of the inverted repeats from the yeast 2 micrometer plasmid.

A sequence of 1019 nucleotides encompassing one of the 600 base inverted repeats and non-repeated flanking regions has been determined in the type A yeast 2 micrometers plasmid cloned in pMB9. Methods are described for applying the Maxam-Gilbert sequencing procedure to DNA fragments labelled at the 3'-end using a T4-polymerase exchange/repair reaction and for sequencing 5'-end labelled fragments using dideoxy-nucleotides as chain terminators in the presence of E. coli DNA polymerase (nach Klenow). A notable feature of the sequence is its unusual content of symmetry elements. In one region of 140 nucleotides, 137 are involved in a complex arrangement of direct and inverted repeats linked by palindromic sequences.