Biomedical subjects
D Sherratt
Publications and source records attributed to D Sherratt.
Determinants of selectivity in Xer site-specific recombination.
A remarkable property of some DNA-binding proteins that can interact with and pair distant DNA segments is that they mediate their biological function only when their binding sites are arranged in a specific configuration. Xer site-specific recombination at natural plasmid recombination sites (e.g., cer in ColE1) is preferentially intramolecular, converting dimers to monomers. In contrast, Xer recombination at the Escherichia coli chromosomal site dif can occur intermolecularly and intramolecularly. Recombination at both types of site requires the cooperative interactions of two related recombinases, XerC and XerD, with a 30-bp recombination core site. The dif core site is sufficient for recombination when XerC and XerD are present, whereas recombination at plasmid sites requires approximately 200 bp of adjacent accessory sequences and accessory proteins. These accessory factors ensure that recombination is intramolecular. Here we use a model system to show that selectivity for intramolecular recombination, and the consequent requirement for accessory factors, can arise by increasing the spacing between XerC- and XerD-binding sites from 6 to 8 bp. This reduces the affinity of the recombinases for the core site and changes the geometry of the recombinase/DNA complex. These changes are correlated with altered interactions of the recombinases with the core site and a reduced efficiency of XerC-mediated cleavage. We propose that the accessory sequences and proteins compensate for these changes and provide a nucleoprotein structure of fixed geometry that can only form and function effectively on circular molecules containing directly repeated sites.
Effects of Holliday junction position on Xer-mediated recombination in vitro.
Site-specific recombination mediated by XerC and XerD functions in the segregation of circular replicons in Escherichia coli. A key feature of most models of recombination for the family of recombinases to which XerC and XerD belong is that a Holliday junction forms at the position of the first pair of recombinase-mediated strand exchanges and then branch migrates 6-8 bp to the position of the second pair of strand exchanges. We have tested this hypothesis for Xer recombination by studying the effects of junction position on XerC-mediated strand exchange in vitro. Recombination of synthetic Holliday junction substrates in which junction mobility was constrained to a region extending over or removed away from the normal cleavage and exchange point was analysed. All substrates undergo strand cleavage at the normal position. We infer that the Holliday junction need not be at this position during strand cleavage and exchange. With substrates in which the Holliday junction is constrained to a region away from the XerC-mediated cleavage point, strand exchange generates products with the predicted mispaired bases.
The sss gene product, which affects pyoverdin production in Pseudomonas aeruginosa 7NSK2, is a site-specific recombinase.
Pyoverdin production by Pseudomonas aeruginosa strain 7NSK2 was induced by Zn(II) in the presence of iron. A mutant was isolated in which Zn(II) no longer induced pyoverdin production. The sss gene which was inactivated in this mutant was cloned and sequenced. Its protein sequence showed 50% identity to the XerC protein of Escherichia coli, which is a member of the lambda integrase family of site-specific recombinases. An open reading frame was found upstream of sss whose protein sequence showed strong identity to DapF, the diaminopimelate epimerase. In E. coli, xerC is part of a multicistronic unit that also contains dapF. The sss gene of P. aeruginosa could restore site-specific recombination at cer in an E. coli xerC mutant and the E. coli xerC gene could complement a genomic sss mutation in P. aeruginosa.
Escherichia coli XerC recombinase is required for chromosomal segregation at cell division.
XerC is a site-specific recombinase of the bacteriophage lambda integrase family that is encoded by xerC at 3700 kbp on the genetic map of Escherichia coli. The protein was originally identified through its role in converting multimers of plasmid ColE1 to monomers; only monomers are stably inherited. Here we demonstrate that XerC also has a role in the segregation of replicated chromosomes at cell division. xerC mutants form filaments with aberrant nucleotides that appear unable to partition correctly. A DNA segment (dif) from the replication terminus region of the E. coli chromosome binds XerC and acts as a substrate for XerC-mediated site-specific recombination when inserted into multicopy plasmids. This dif segment contains a region of 28 bp with sequence similarity to the crossover region of ColE1 cer. The cell division phenotype of xerC mutants is suppressed in strains deficient in homologous recombination, suggesting that the role of XerC/dif in chromosomal metabolism is to convert any chromosomal multimers (arising through homologous recombination) to monomers.
Identification of functional regions of the colicinogenic plasmid ColA.
ColA is a colicinogenic plasmid of 6.72 kb. It is compatible with ColE1 but not with ColK. Transposon insertion mutagenesis as well as complementation studies have been carried out to investigate the location of the various functional regions of this plasmid. Four independent ColA::Tn1 and one ColA::Tn3 plasmids were isolated and the locations of insertions were determined. From these plasmids, six different deletion mutants were constructed. In addition, various restriction fragments of ColA have been cloned into pUC8 to carry out complementation studies. We have thus confirmed the location of the DNA regions involved in colicin production, colicin release and immunity function. The DNA region involved in conjugal mobility promoted by R64 drd11 has been identified and we have demonstrated that the ColE1 mobility proteins can act in trans on the bom (basis of mobility) site of ColA. The location of this site, as well as the region involved in stable maintenance of ColA, have also been determined. These results are discussed with regard to the homology in nucleotide sequence between ColA and ColE1.
Analysis of Tn7 transposition.
Five gene products required for Tn7 transposition were identified using genetic complementation tests. Four of these (tnsA, tnsB, tnsC and tnsD) are essential for insertion into the attachment site, whereas the fifth (tnsE) is required for transposition to plasmids which lack this site. tnsD is not required for transposition to plasmids lacking the attachment site. This analysis used a chloramphenicol-resistant 'mini' transposon containing Tn7 termini but no complete Tn7 gene product and several compatible expression vectors containing cloned Tn7 fragments. The use of transcriptional and translational fusions allowed the identification of two promoters (P1 and P2) at the righthand end of the transposon and indicated that at least tnsA, tnsB and tnsC are translated in the same direction. Expression from P1 appears to be repressed by tnsB.
Multimer resolution systems of ColE1 and ColK: localisation of the crossover site.
We have identified and characterised a stability function encoded by the high copy plasmid ColK. The function is analogous to ColE1 cer and maximises stability by maintaining plasmids in the monomeric state. In vivo recombination between cer and ckr (which share more than 90% homology at the DNA sequence level) produced a functional hybrid. Sequence analysis of hybrids indicates that recombination involving cer and ckr is site-specific and occurs within a 35 bp region of DNA which contains palindromic symmetry.
Transposition and transposition immunity of transposon Tn3 derivatives having different ends.
Novel Tn1/3 derivatives that contained either two left- or two right-hand ends of the transposon were constructed in a small plasmid. Both transposed at reasonable frequencies to give normal transposition products, suggesting that only the 38-bp inverted repeats of Tn3 are essential for transposition. Plasmids containing transposon derivatives with only one end (either left or right) undergo transposase-dependent transposition between replicons at much lower frequencies, resulting in co-integrate molecules in which there is no substantial duplication of transposon DNA and that appear to be simple fusions of the two plasmids. Both the right and left halves of the transposon are separately able to confer transposition immunity to the plasmid, this immunity being inseparably linked to transposition proficiency and specificity.
Transposon Tn1 intra-molecular transposition.
A system for the direct selection of intra- and inter-molecular transposition events has been used to show that intra-molecular transposition of Tn1 generates deletions and inversions and requires the tnpA but not the tnpR gene product, as predicted by current models of transposition. Intra-molecular Tn1 transposition is much less limited by 'transposition immunity' than inter-molecular transposition, and occurs at frequencies comparable to those for inter-molecular transposition. The selection system, which uses the bacteriophage lambda cI- PR region as a target can be used to select, quantify, and characterize any spontaneous or induced mutations.
Site-specific recombination in transposition and plasmid stability.
Explore the source record for details and available documents.
Plasmid ColE1 conjugal mobility: the nature of bom, a region required in cis for transfer.
Conjugal mobility of ColE1 and related plasmids is promoted by a wide range of conjugative plasmids. ColE1 produces trans-acting products and has a region required in cis (bom ; basis of mobility) for such mobility. Here we show that plasmid pBR322 contains a functional bom sequence located within a 141 bp HhaI fragment. This bom region is functional for conjugation promoted by several different conjugative plasmids and is highly conserved in ColE1 and contains nic the putative origin of transfer. The orientation and position of bom with respect to the ColE1 vegetative origin of replication can be changed without affecting the frequency of conjugal mobility promoted by R64drd11.
Transposon-specified site-specific recombination.
Cointegrate DNA molecules containing two copies of a transposable element appear to be intermediates in the transposition process. These structures are resolved by site-specific recombination to yield the normal end products of transposition. The transposable element gamma delta (Tn1000) synthesizes a product interchangeable with the Tn1/3tnpR protein in promoting Tn1/3 site-specific recombination. These data support the hypothesis that cointegrates containing directly repeated copies of Tn1/3 are obligatory intermediates in interreplicon transposition of Tn1/3. In addition, we show here that the reaction is independent of the element-encoded tnpA gene product. Tn501, which specifies mercury resistance, also produces cointegrates as intermediates in interreplicon transposition. The appearance of Tn501-specified recombination activity that can act on these cointegrates requires growth of cells in the presence of Hg2+.
Site-specific recombination.
Explore the source record for details and available documents.
Structure and function of plasmid ColK.
Explore the source record for details and available documents.
Transposon-specified, site-specific recombination systems.
Explore the source record for details and available documents.
Trans-complementable copy-number mutants of plasmid ColE1.
Plasmid ColE1, like many other small non-conjugative plasmids, is present in multiple copies (about 15 per chromsome equivalent) in Escherichia coli cells. Because of their high copy number, the replication of such plasmids has been described as 'relaxed', even though there is good evidence that it is strictly controlled: ColE1 derivatives have characteristic but different copy numbers and ColE1 copy-number mutants have been characterised. No plasmid-specified protein is essential for the replication of ColE1 and related plasmids, as extensive replication can occur in chloramphenicol-treated cells, in plasmid-free chloramphenicol-treated cells transfected with a hybrid ColE1/phage replicon and in vitro in extracts derived from plasmid-free cells. Nevertheless, it is possible that a plasmid-specified protein is involved in ColE1 replication control in viable cells. Here we show that deletion of a given non-essential region from ColE1-like plasmids results in a raised copy number. Such plasmids are stably maintained and have their copy number returned to normal when a complementing plasmid is present in the same cell, indicating that a plasmid-specified diffusible gene product regulates the plasmid content of ColE1-containing cells. Deletion of the equivalent region from the cloning vector pBR322 gives a derivative which has a raised copy number and which has also lost its origin for conjugal transfer; unlike pBR322, it cannot be mobilised.
Dissection of the transposition process: a transposon-encoded site-specific recombination system.
Explore the source record for details and available documents.