ORCID Profile
0000-0003-1776-0738
Current Organisations
New South Wales Department of Health
,
Eriter Consulting
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Publisher: American Society for Microbiology
Date: 09-1988
DOI: 10.1128/JB.170.9.4083-4090.1988
Abstract: 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.
Publisher: Elsevier BV
Date: 12-1989
DOI: 10.1016/0378-1119(89)90479-4
Abstract: 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.
Publisher: Elsevier BV
Date: 07-1996
Abstract: The recent discovery of the Bacillus subtilis plasmid terminator TerLS20 with bidirectional fork arrest activity has provided the opportunity to probe further the structural and functional features of B. subtilis replication terminators in general. The minimal TerI and TerLS20 terminators each comprise two 13 nt segments flanking a central trinucleotide, which is almost completely conserved in all terminators. It corresponds to the region of overlap of the two RTP binding sites (A and B) on the DNA. It has been shown that, despite this conservation, considerable variation in this trinucleotide region still allows fork arrest activity. Thus, the productive interaction of the RTP dimers, which presumably occurs in the vicinity of this trinucleotide region, is not dependent upon stringently defined contacts with the bases in this region. A completely synthetic and highly symmetrical terminator was constructed by replacing the 13 nt segment of the A site of TerI with an opposed segment identical to that in the B site. The efficient bidirectional activity of this new terminator, TerSymB, established more firmly the need for two opposed RTP binding sites in a functional terminator. TerSymB was used to investigate the effect of sequence deviation in one of the 13 nt segments, from that in the B site, on bidirectionality of the terminator. It was found that the deviations introduced converted the terminator significantly towards polarity of action. The partial symmetry within each of the 13 nt segments of TerSymB, and the presumed recognition of this symmetry in the binding of a symmetrical dimer of RTP to each overlapping site, suggest that the bound dimers are centred over positions in the DNA sequence separated by 15 nt. This separation distance has been used in conjunction with the mode of binding of RTP to DNA proposed by Bussiere et al., based on their crystal structure for RTP, to model the interaction of the two dimers of RTP with unbent B-form DNA. Increased separation of the two binding sites of TerSymB was performed by inserting an extra three, seven or ten nucleotides centrally within the TerSymB sequence. The effects of these insertions on RTP binding and fork arrest activity were consistent with the proposed positioning of the RTP dimers within the terminator sequence, and interaction between the dimers bound to TerSymB. A model to account for the generation of RTP-terminator complexes with bidirectional or polar fork arrest activity utilising TerSymB or TerI-VI is presented.
Publisher: Elsevier BV
Date: 09-1983
DOI: 10.1016/0378-1119(83)90133-6
Abstract: DNA from the Bacillus subtilis 168 prototroph, SB19, was partially cleaved with MboI and cloned into the BamHI site of the Escherichia coli cosmid vector, pHC79. [3H]thymine-labelled DNA from the replication terminus region of the B. subtilis dna-1 chromosome was used to identify, by hybridization, clones harboring recombinant cosmids carrying inserts from the terminus region. Restriction maps have been constructed for two such cosmids carrying overlapping DNA inserts that cover or extend into four of the previously identified five SalI fragments which are replicated last. The composite map of the cloned region, together with the available data on the replication order of fragments within it, is consistent with its replication being achieved by the unidirectional movement of a fork through it and towards the late replicating 16.2-kb SalI fragment. Most, if not all, of the terminus sequences in at least one of the recombinant cosmids are missing from a viable strain of B. subtilis that carries a deletion in the SP beta-gltA region of the chromosome.
Publisher: Elsevier BV
Date: 12-1984
DOI: 10.1016/0378-1119(84)90045-3
Abstract: The library of Bacillus subtilis DNA previously cloned in the cosmid vector pHC79 has been screened for the replication terminus region using a higher level of terminus probe. 24 of 48 recombinant cosmids which gave a positive response had restriction fragment compositions consistent with their inserts originating from or extending into the terminus region for which a 150-kb restriction map has already been constructed (Weiss and Wake, 1983). DNA spanning terC, the site of termination, appears to be missing from the library, although DNA to either side of terC has been cloned. A detailed analysis of four of the newly identified recombinant cosmids has confirmed most of the previous 150-kb map and allowed it to be extended to 180 kb. Physical linkage of the two cosmid inserts that most closely approach terC on each side has been demonstrated. The location of the genetic marker gltA and the orientation of the restriction map relative to the genetic map of the B. subtilis chromosome have also been established.
Publisher: Elsevier BV
Date: 03-1999
Abstract: DNA replication fork arrest during the termination phase of chromosome replication in Bacillus subtilis is brought about by the replication terminator protein (RTP) bound to specific DNA terminator sequences (Ter sites) distributed throughout the terminus region. An attractive suggestion by others was that crucial to the functioning of the RTP-Ter complex is a specific interaction between RTP positioned on the DNA and the helicase associated with the approaching replication fork. In support of this was the behaviour of two site-directed mutants of RTP. They appeared to bind Ter DNA normally but were ineffective in fork arrest as ascertained by in vitro Escherichia coli DnaB helicase and replication assays. We describe here a system for assessing the fork-arrest behaviour of RTP mutants in a bona fide in vivo assay in B. subtilis. One of the previously studied mutants, RTP.Y33N, was non-functional in fork arrest in vivo, as predicted. But through extensive analyses, this RTP mutant was shown to be severely defective in binding to Ter DNA, contrary to expectation. Taken in conjunction with recent findings on the other mutant (RTP.E30K), it is concluded that there is as yet no substantive evidence from the behaviour of RTP mutants to support the RTP-helicase interaction model for fork arrest. In an extension of the present work on RTP.Y33N, we determined the dissociation rates of complexes formed by wild-type (wt) RTP and another RTP mutant with various terminator sequences. The functional wtRTP-TerI complex was quite stable (half-life of 182 minutes), reminiscent of the great stability of the E. coli Tus-Ter complex. More significant were the exceptional stabilities of complexes comprising wtRTP and an RTP double-mutant (E39K.R42Q) bound to some particular terminator sequences. From the measurement of in vivo fork-arrest activities of the various complexes, it is concluded that the stability (half-life) of the whole RTP-Ter complex is not the overriding determinant of arrest, and that the RTP-Ter complex must be actively disrupted, or RTP removed, by the action of the approaching replication fork.
Publisher: Elsevier BV
Date: 08-1994
Abstract: The 47 bp DNA replication terminator (IRI) of Bacillus subtilis, contains two binding sites, A and B, for the replication terminator protein (RTP). Each site binds a dimer of RTP. Removal of the first two base-pairs (bp 1-2) from IRI completely destroyed in vivo terminator (fork arrest) function and was accompanied by loss of RTP binding to the A site, which is distal to the approaching fork that is arrested. Removal of base-pairs 34 to 47 from the other end, proximal to the approaching fork, lowered in vivo function to approximately 50% of the complete IRI. RTP binding appeared to be largely unaffected. Terminator function remained at the approximately 50% level with further deletions that proceeded as far as to include base-pair 28 and RTP binding remained largely unaffected. Removal of more of the sequence beyond base-pair 27 and into the region that makes extensive contact with RTP resulted in a further impairment to in vivo function, and caused altered RTP binding. The base-pairs 1 to 24 segment retained only 16% fork arrest activity and the effect on RTP binding was largely evidenced by an elimination of the ability of this extensively truncated sequence to fill the B site alone. The behaviour of the various terminator deletions emphasize the importance of the previously defined RTP-DNA contacts which allow the binding of RTP to the two overlapping sites, A and B, of IRI for terminator function. A comparison of the affinities of selected truncated terminators for RTP raises the possibility that the overall affinity of RTP for its DNA terminator is not the sole determinant of terminator function.
Publisher: Wiley
Date: 11-1993
DOI: 10.1111/J.1365-2958.1993.TB00947.X
Abstract: The interaction between the DNA replication terminator, IRI, of Bacillus subtilis and its cognate replication terminator protein (RTP) has been examined by the technique of missing nucleoside interference (MNI). IRI contains two adjacent binding sites (A and B) for RTP dimers. The B site is proximal to the replication fork arrest site. The present results have shown that nucleoside contacts with RTP in the two sites are very different. There are more extensive contacts of nucleosides in both strands of the B site with RTP compared with the A site. The data also strongly suggest that filling by RTP of the B site occurs first and is needed for subsequent co-operative filling of an overlapping A site. The A site alone binds RTP poorly. The findings are consistent with interaction occurring between RTP dimers bound to adjacent sites of IRI, which would explain why RTP bound to the B site alone cannot cause replication fork arrest.
Publisher: Wiley
Date: 03-1996
DOI: 10.1111/J.1365-2958.1996.TB02474.X
Abstract: We have sequenced and analysed a 3.1 kb fragment of the 55 kb endogenous Bacillus subtilis plasmid pLS20 containing its replication functions. Just outside the region required for autonomous replication, a segment of 18 bp was identified as being almost identical to part of the major B. subtilis chromosomal replication terminator. Here, we demonstrate that this segment is part of a functional replication terminator. This newly identified element, designated TerLS20, is the first replication terminator identified on a theta plasmid from a Gram-positive bacterium. TerLS20 is distinct from other known replication terminators in the sense that it is functional in both orientations. The region required for bipolar functionality of TerLS20 was delineated to a sequence of 29 bp, which is characterized by an imperfect dyad symmetry.
Publisher: American Society for Microbiology
Date: 06-1989
DOI: 10.1128/JB.171.6.3564-3567.1989
Abstract: 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.
Publisher: Microbiology Society
Date: 10-1987
DOI: 10.1099/00221287-133-10-2707
Abstract: DNA from each of two specialized transducing lambda phage, lambda dcysJIHD and lambda cysJ, has been analysed by heteroduplex mapping. The segment of the Escherichia coli chromosome carried by lambda dcysJIHD was shown to be large, approximately 18 kb in length, and to replace a large length of lambda DNA, approximately 11 kb, which includes the genes for integration and recombination. Thus lambda dcysJIHD is a bio-type transducing phage. lambda cysJ was shown to have lost very little lambda DNA and to carry about 8 kb of bacterial DNA. Sites for several restriction endonucleases were mapped in the DNA from each phage and cloning experiments located some of the genes of the cluster in relation to the restriction map. Cysteine regulation of the cloned cysJ and cysD genes was shown as well as cysteine regulation of beta-galactosidase in some constructs. The direction of transcription of the cysD gene was established, and from physical evidence the size of the 'silent section' between the cysH and cysD genes was estimated to be at least 11 kb.
Publisher: Elsevier BV
Date: 10-1992
DOI: 10.1016/0022-2836(92)90214-5
Abstract: 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.
Publisher: Oxford University Press (OUP)
Date: 1987
Abstract: 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.
Publisher: Elsevier BV
Date: 09-1993
DOI: 10.1016/0378-1119(93)90508-Z
Abstract: One of two putative sigma A promoters identified previously in the region immediately upstream from the rtp gene (encoding the replication terminator protein) [Smith and Wake, J. Bacteriol. 170 (1988) 4083-4090] has been shown by transcription start point (tsp) mapping to be the functional rtp promoter. In these tsp mapping experiments, it was observed that the level of mRNA from this promoter, Prtp, was increased by a factor of 30 in the absence of the replication terminator protein (RTP), consistent with the autoregulation of rtp at the level of transcription. In vitro transcription from Prtp by sigma A RNA polymerase has been shown to be specifically repressed by RTP. A Prtp-spoVG-lacZ fusion was inserted into the chromosome of a strain in which RTP production was inducible by IPTG. Addition of IPTG to cultures of the new strain lowered beta Gal production by a factor of at least four. It is concluded that rtp is autoregulated in vivo at the level of transcription.
Publisher: Elsevier BV
Date: 1985
DOI: 10.1016/0378-1119(85)90198-2
Abstract: 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.
Publisher: Elsevier BV
Date: 11-1991
DOI: 10.1016/0022-2836(91)90206-L
Abstract: 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.
Location: No location found
Location: Australia
No related grants have been discovered for Mark Smith.