A sequence similarity between proteins involved in initiation and termination of bacterial chromosome replication.
Explore the source record for details and available documents.
Amino Acid Sequence↗
Biomedical subjects
R G Wake
Publications and source records attributed to R G Wake.
Variations and coding features of the sequence spanning the replication terminus of Bacillus subtilis 168 and W23 chromosomes.
In a comparative study of the sequences of the 3-kb regions of DNA spanning the replication terminus, terC, of Bacillus subtilis strains 168 and W23, it was found that the latter contained an insertion of a large open reading frame (ORF405) whose translated protein product is a member of the cytochrome P-450 family. The insertion was about 34 nucleotides upstream from a putative promoter for the rtp gene. The sequenced regions contained a number of other ORFs. The translation product of one (ORF238) is a member of a previously identified oxidoreductase superfamily. The translation product of another (ORF257) is significantly similar to the proC product of Escherichia coli, but this ORF does not code for a functional proC product of B. subtilis.
Amino Acid Sequence↗
Chromosome strand segregation during sporulation in Bacillus subtilis.
After the initiation of spore formation in Bacillus subtilis, the products of the final round of DNA replication segregate into two cells, i.e. the prespore and the mother cell. The prespore, which is known to contain a single completed chromosome, develops into a mature endospore which can be readily separated from mother cells and non-sporulating cells on the basis of its resistance properties. We have used a procedure originally developed to label the terminus region of the B. subtilis chromosome to specifically label the newly synthesized strands of DNA during the final round of DNA replication before sporulation. We have purified prespore DNA and used strand-specific probes to measure the radioactivity incorporated. The results show that the sister chromosomes segregate at random into the prespore. This result has implications for the segregation of chromosomes during vegetative growth and for the generation of cellular asymmetry during sporulation.
Bacillus subtilis↗
Location of the dihydroorotase domain within trifunctional hamster dihydroorotate synthetase.
Mammalian dihydroorotase (DHOase, EC 3.5.2.3) is part of a trifunctional protein, dihydroorotate synthetase which catalyzes the first three reactions of de novo pyrimidine biosynthesis. We have subcloned a portion of the cDNA from the plasmid pCAD142 and obtained a nucleotide sequence which extends 2.1 kb in the 5' direction from the sequence encoding the aspartate transcarbamoylase (ATCase) domain at the 3'-end of the cDNA. The DHOase and ATCase domains have been purified from an elastase digest of the trifunctional protein and subjected to amino acid (aa) sequencing from their N termini. The sequence of the N-terminal 24 aa of the DHOase domain has been obtained and aligned with the cDNA sequence. The C-terminal residues of the DHOase domain have been identified as Leu followed by Val which, when taken with partial sequences of the CNBr fragments of this domain, defines the coding sequence of the active, globular DHOase domain released by proteolysis. Prediction of protein secondary structure from the deduced aa sequence showed that the DHOase domain (Mr 37,751) is separated from the C-terminal ATCase domain (Mr 34,323) by a bridging sequence (Mr 12,532) consisting of multiple beta-turns.
Amino Acid Sequence↗
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.
Bacillus subtilis↗
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.
Bacillus subtilis↗
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.
Bacillus subtilis↗
Cloning and expression of a Bacillus subtilis division initiation gene for which a homolog has not been identified in another organism.
The Bacillus subtilis 168 division initiation genes defined by the temperature-sensitive mutations ts-1 and ts-12 were cloned into a 10.5-kilobase EcoRI fragment of DNA in the lambda EMBL4 vector. The two genes were separated by approximately 3 kilobases. The gene in which the ts-1 mutation resides was shown to be the same as the B. subtilis homolog of the Escherichia coli ftsZ gene. The other gene was named divIB. It showed no homology to any previously identified gene and coded for a protein of 30.1 kilodaltons which was probably membrane bound.
Amino Acid Sequence↗
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).
Amino Acid Sequence↗
A protein involved in termination of chromosome replication in Bacillus subtilis binds specifically to the terC site.
The small basic protein encoded by the open reading frame adjacent to the terC site in the Bacillus subtilis chromosome and previously implicated in termination of the replication process was purified. Band retardation assays established that this protein (now called the replication terminator protein, encoded by the rtp gene) binds specifically to a 209-base-pair fragment of DNA within which terC is located.
Bacillus subtilis↗
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.
Bacillus subtilis↗
DNA sequence requirements for replication fork arrest at terC in Bacillus subtilis.
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.
Amino Acid Sequence↗
Sequence features of the replication terminus of the Bacillus subtilis chromosome.
The sequence of 1267 nucleotides spanning the replication terminus, terC, of the Bacillus subtilis 168 chromosome has been determined. The site of arrest of the clockwise fork, which defines terC, has been localized to a 30-nucleotide portion (approximately) within this sequence. The arrest site occurs in an A + T-rich region between two open reading frames and very close to one of two imperfect inverted repeats (47-48 nucleotides each) which are separated by 59 nucleotides. The closeness of approach of the arrested clockwise fork to the first imperfect inverted repeat encountered in this region raises the possibility of a role for the inverted repeats in the mechanism of fork arrest.
Amino Acid Sequence↗
Breakdown and quantitation of the forked termination of replication intermediate of Bacillus subtilis.
Using a procedure that minimizes shear forces, the BamHI-derived forked termination of replication intermediate of Bacillus subtilis, called band I DNA, can be extracted with little or no accompanying band II DNA. It has been shown that band II DNA is a product of band I breakdown. Nuclease P1-mediated breakdown of the forked band I DNA proceeds in two steps. The first causes the release of one of the arms as band II DNA; in the second step, the remaining arm is cleaved away to yield the free stem. It is concluded that band I represents the primary termination of replication intermediate. A quantitative assessment of the level of band I in DNA from cells of the merodiploid strain, GSY1127, growing at different rates has been made. For cells grown in a minimal medium, at least, the experimentally measured level of band I is of the order (approx. 60%) of that predicted for a complete block to movement of the clockwise fork at the replication terminus, terC.
Bacillus subtilis↗
The normal replication terminus of the Bacillus subtilis chromosome, terC, is dispensable for vegetative growth and sporulation.
The Bacillus subtilis strains CU1693, CU1694 and CU1695 were shown by hybridization analysis to carry large deletions of the terminus region that originated within discrete fragments of the SP beta prophage genome. The absence of terC in CU1693 was demonstrated definitively by the identification of a novel junction fragment comprising SP beta DNA and DNA that lies on the other side of terC in the parent strain. This represented the deletion of approximately 230 kb of CU1693 DNA, with the removal of approximately 150 kb to the left of terC and approximately 80 kb to the right of terC. The lack of hybridization of CU1694 and CU1695 DNA to cloned DNA carrying the terC sequence and to cloned DNAs flanking terC suggested that terC is absent from the chromosome of each of these strains also, and that the deletions in CU1694 and CU1695 extend beyond the segment of the terminus region that has been mapped and cloned. The normal growth rate and morphology of CU1693, CU1694 and CU1695 relative to the parent strain when grown in complex medium indicated dispensability of terC for vegetative growth and division. B. subtilis SU153 was constructed using a specific deletion-insertion vector that was designed to effect the deletion of 11.2kb of DNA spanning terC, with the removal of approximately 9.7kb to the left of terC and approximately 1.kb to the right of terC. This manipulation did not introduce any readily detectable auxotrophic requirement. Physiological characterization of SU153 confirmed the dispensability of terC for vegetative growth and cell division, and also established the lack of requirement of terC for the specialized cell division that is associated with formation of the bacterial endospore.
Bacillus subtilis↗
An immobilized fork as a termination of replication intermediate in Bacillus subtilis.
The structure of a DNA intermediate associated with termination of chromosome replication in Bacillus subtilis and derived from a unique BamHI 24.8 X 10(3) base-pair (bp) region of the chromosome has been investigated. The intermediate has properties expected for a forked structure. Gel electrophoresis followed by Southern transfer and hybridization to cloned DNA has shown it to comprise single strands of 15.4 X 10(3) bp and 24.8 X 10(3) bp, in approximately equimolar amounts. After purification away from the bulk of chromosomal DNA, electron microscopy of the intermediate established that 15% of the DNA was present as branched molecules and a significant proportion (11 of 31) of these contained two arms of matching length. The average dimensions (best estimates) of this unique class of Y-shaped molecule were 9.5(+/- 0.3) X 10(3), 15.1(+/- 0.4) X 10(3) and 24.6 24.6(+/- 0.6) X 10(3) bp for the stem, arms and end-to-end length, respectively. These values are consistent with the single strand composition of the intermediate as found. Furthermore, hybridization of the single strands to DNA from known locations within the BamHI 24.8 X 10(3) bp region has established the orientation of the forked intermediate relative to the genetic map. The intermediate presumably reflects the immobilization of the clockwise replication fork within the 24.8 X 10(3) bp region, at a location approximately 15.4 X 10(3) bp from the right end.
Bacillus subtilis↗
Nonrandom cosmid cloning and prophage SP beta homology near the replication terminus of the Bacillus subtilis chromosome.
From a library of Bacillus subtilis DNA cloned with the Escherichia coli cosmid vector pHC79, 85 recombinant cosmids containing DNA from near the replication terminus, terC, were identified. The DNA inserts of these cosmids were confined to three regions of a 350-kilobase segment of the chromosome extending from the left end of the SP beta prophage to approximately 75 kilobases on the right of terC. All B. subtilis genes known to reside in this segment, as well as the portion of the SP beta prophage that is expressed early in the lytic cycle of the phage, appeared to be absent from the library. A region of SP beta homology distinct from the prophage and just to the left of terC was identified.
Bacillus subtilis↗
Cloning and localization of the Bacillus subtilis chromosome replication terminus, terC.
A 10.9-kb segment of the Bacillus subtilis 168 chromosome has been cloned in an Escherichia coli plasmid and shown to contain terC (the replication terminus of the chromosome). The terC-containing portion of this plasmid has been subcloned within each of two overlapping fragments of DNA, 1.75 and 1.95 kb, again in E. coli plasmids. These have afforded a more precise definition of the location of terC in the B. subtilis chromosome and provided material for a detailed analysis of the structure and functioning of this site.
Bacillus subtilis↗