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D J Sherratt

Publications and source records attributed to D J Sherratt.

At least 19 recordsLinked to original sources
PubMed1997

Crystal structure of the site-specific recombinase, XerD.

The structure of the site-specific recombinase, XerD, that functions in circular chromosome separation, has been solved at 2.5 A resolution and reveals that the protein comprises two domains. The C-terminal domain contains two conserved sequence motifs that are located in similar positions in the structures of XerD, lambda and HP1 integrases. However, the extreme C-terminal regions of the three proteins, containing the active site tyrosine, are very different. In XerD, the arrangement of active site residues supports a cis cleavage mechanism. Biochemical evidence for DNA bending is encompassed in a model that accommodates extensive biochemical and genetic data, and in which the DNA is wrapped around an alpha-helix in a manner similar to that observed for CAP complexed with DNA.

Amino Acid Sequence↗
PubMed1997

Direct interaction of aminopeptidase A with recombination site DNA in Xer site-specific recombination.

Xer site-specific recombination at ColE1 cer converts plasmid multimers into monomers, thus ensuring the heritable stability of ColE1. Two related recombinase proteins, XerC and XerD, catalyse the strand exchange reaction at a 30 bp recombination core site. In addition, two accessory proteins, PepA and ArgR, are required for recombination at cer. These two accessory proteins are thought to act at 180 bp of accessory sequences adjacent to the cer recombination core to ensure that recombination only occurs between directly repeated sites on the same molecule. Here, we demonstrate that PepA and ArgR interact directly with cer, forming a complex in which the accessory sequences of two cer sites are interwrapped approximately three times in a right-handed fashion. We present a model for this synaptic complex, and propose that strand exchange can only occur after the formation of this complex.

Amino Acid Sequence↗
PubMed1997

Xer recombination in Escherichia coli. Site-specific DNA topoisomerase activity of the XerC and XerD recombinases.

Xer site-specific recombination functions in maintaining circular replicons in the monomeric state in Escherichia coli. Two recombinases of the bacteriophage lambda integrase family, XerC and XerD, are required for recombination at the chromosomal site, dif, and at a range of plasmid-borne sites. Xer recombination core sites contain the 11-base pair binding sites for each recombinase separated by a 6 to 8-base pair central region. We report that both XerC and XerD act as site-specific type I topoisomerases by relaxing supercoiled plasmids containing a dif site. Relaxation by either XerC or XerD occurs in the absence of the partner recombinase and requires only a single recombination core site. XerC or XerD relaxation activities are completely inhibited by the addition of the partner recombinase, providing that the DNA recognition sequence for the inhibiting partner is present.

Binding Sites↗
PubMed1997

Effect of DNA topology on Holliday junction resolution by Escherichia coli RuvC and bacteriophage T7 endonuclease I.

Holliday junctions are key intermediates in homologous genetic recombination. Their resolution requires specialised nucleases that nick pairs of strands at the junction point, leading to the separation of mature recombinants. Resolution occurs in either of two orientations, according to which DNA strands are cut. We show that DNA topology can determine the efficiency and outcome of a recombination reaction. Using two Holliday junction resolvases, Escherichia coli RuvC protein and T7 endonuclease I, we observed that supercoiled figure-8 DNA molecules containing Holliday junctions were resolved with a specific orientation bias, and that this bias was reversed by the presence of a topological tether (catenation). In contrast, when all topological constraints were removed by restriction digestion, the recombination intermediates were resolved equally in the two orientations. These results show that topological constraints affecting Holliday junction structure influence the orientation of resolution by cellular resolvases.

Bacterial Proteins↗
PubMed1997

Action of site-specific recombinases XerC and XerD on tethered Holliday junctions.

In Xer site-specific recombination, two related recombinases, XerC and XerD, mediate the formation of recombinant products using Holliday junction-containing DNA molecules as reaction intermediates. Each recombinase catalyses the exchange of one pair of specific strands. By using synthetic Holliday junction-containing recombination substrates in which two of the four arms are tethered in an antiparallel configuration by a nine thymine oligonucleotide, we show that XerD catalyses efficient strand exchange only when its substrate strands are 'crossed'. XerC also catalyses very efficient strand exchange when its substrate strands are 'crossed', though it also appears to be able to mediate strand exchange when its substrate strands are 'continuous'. By using chemical probes of Holliday junction structure in the presence and absence of bound recombinases, we show that recombinase binding induces unstacking of the bases in the centre of the recombination site, indicating that the junction branch point is positioned there and is distorted as a consequence of recombinase binding.

Bacterial Proteins↗
PubMed1997

Pentapeptide scanning mutagenesis: random insertion of a variable five amino acid cassette in a target protein.

A new insertion method for probing protein functional organization was developed. The method relies on the random insertion of transposon Tn 4430 and subsequent in vitro deletion of the bulk of the transposon after which a 15 bp insertion remains within the target gene. This results in pentapeptide insertions randomly distributed in the target protein. Characterization of 23 pentapeptide insertions in TEM-1beta-lactamase demonstrated the utility of the method. The phenotypes associated with the mutated beta-lactamase proteins equated both with the sorts of local peptide structures in which the pentapeptide insertions occurred and their position in the three-dimensional structure of the enzyme.

DNA Transposable Elements↗
PubMed1997

Topological selectivity in Xer site-specific recombination.

The product topology of Xer-mediated site-specific recombination at plasmid sites has been determined. The product of deletion at pSC101 psi is a right-handed antiparallel 4-noded catenane. The ColE1 cer deletion product has an identical topology, except that only one pair of strands is exchanged. These specific product topologies imply that the productive synaptic complex and the strand exchange mechanism have fixed topologies. Further analysis suggests that synapsis traps exactly three negative supercoils between recombining sites, and that strand exchange introduces a further negative topological node in the deletion reaction. We present a model in which the requirement for a specific synaptic stucture, with two recombination sites interwrapped around the accessory proteins ArgR and PepA, ensures that recombination only occurs efficiently between directly repeated sites on the same DNA molecule.

DNA Nucleotidyltransferases↗
PubMed1997

Recombinase binding specificity at the chromosome dimer resolution site dif of Escherichia coli.

Xer site-specific recombination functions in Escherichia coli chromosome segregation and cell division apparently by resolving chromosome dimers, which arise through homologous recombination, to monomers. Xer recombination requires two closely related site-specific recombinases, XerC and XerD, which bind cooperatively to the recombination site dif and catalyse separate pairs of strand exchanges. The dif site is an imperfect palindrome whose left and right halves are bound by XerC and XerD, respectively. By using variant dif sites in which the symmetry between the XerC and XerD binding sites was increased incrementally, the determinants in the dif site that specifically direct binding of XerC and XerD to their cognate sites were elucidated. The primary specificity nucleotides in the XerC and XerD binding sites were identified and their relative contributions to specificity assessed. The biological affects of these mutations on site-specific recombination, chromosome segregation and cell division were examined. The specificity determinants are confined to the non-palindromic outer ends of the binding sites. Replacement of the wild-type dif site with mutated dif sites at the normal location in the replication terminus region of the chromosome revealed that the sequence of the dif site can be altered substantially while retaining apparently normal chromosome segregation activity.

Base Sequence↗
PubMed1997

Binding and cleavage of nicked substrates by site-specific recombinases XerC and XerD.

In Xer site-specific recombination two related recombinases, XerC and XerD, catalyse strand cleavage and rejoining reactions at a site, dif, in order to ensure normal chromosome segregation during cell division in Escherichia coli. We have used nicked suicide substrates to trap reaction intermediates and show that XerC cleaves the top strand efficiently while XerD is less efficient at cleaving the bottom strand of dif. Recombinase-mediated cleavage positions are separated by six base pairs and occur at either end of the dif central region adjacent to the recombinase binding sites. XerC can cleave the top strand of dif inefficiently in the absence of its partner recombinase during a reaction that does not require intermolecular synapsis. The presence of a nick in the bottom strand of dif allows cooperative interactions between two XerC protomers bound to adjacent binding sites, suggesting that a conserved interaction domain is present in both XerC and XerD. Cooperativity between two identical recombinase protomers does not occur on un-nicked linear DNA. Ethylation interference footprinting of two XerD catalytic mutant proteins suggests that the conserved domain II arginine from the integrase family RHRY tetrad may make direct contact with the scissile phosphate.

Alkylation↗
PubMed1997

DNA sequence of recombinase-binding sites can determine Xer site-specific recombination outcome.

Xer site-specific recombination functions in the stable inheritance of circular plasmids and bacterial chromosomes. Two related recombinases, XerC and XerD, mediate this recombination, which 'undoes' the potential damage of homologous recombination. Xer recombination on natural plasmid sites is preferentially intramolecular, converting plasmid multimers to monomers. In contrast, recombination at the Escherichia coli recombination site, dif, occurs both intermolecularly and intramolecularly, at least when dif is inserted into a multicopy plasmid. Here the DNA sequence features of a family of core recombination sites in which the XerC- and XerD-binding sites, which are separated by 6 bp, were analysed in order to ascertain what determines whether recombination will be preferentially intramolecular, or will occur both within and between molecules. Sequence changes in either the XerC- or XerD-binding site can alter the recombination outcome. Preferential intramolecular recombination between a pair of recombination sites requires additional accessory DNA sequences and accessory recombination proteins and is correlated with reduced affinities of recombinase binding to recombination core sites, reduced XerC-mediated cleavage in vitro, and an apparent increased overall bending in recombinase-core-site complexes.

Base Sequence↗
PubMed1997

Relating primary structure to function in the Escherichia coli XerD site-specific recombinase.

XerC and XerD are related 298-amino-acid site-specific recombinases, each of which is responsible for the exchange of one pair of strands in Xer recombination. Both recombinases encode functions necessary for sequence-specific DNA-binding, co-operative XerC/D interactions, synapsis and catalysis. These functions were related to the primary amino acid sequence by constructing and analysing internal and C-terminal XerD deletions. An XerD derivative containing residues 1-233 was proficient in specific DNA binding, but did not interact co-operatively with XerC. Deletion of a further five C-terminal amino acids abolished binding to DNA. Proteins deleted for residues 32-88 and for residues 145-159 were deficient in DNA binding. Deletion of residues 244-281, a region containing amino acids necessary for catalysis, gave a protein that bound to DNA. An XerD derivative containing residues 1-268 retained co-operative interactions with XerC; nevertheless, it did not support XerC strand exchange and was defective in XerD catalysis. Residues 1-283 retain a functional catalytic active site, though a protein lacking the five C-terminal amino acids was still unable to mediate normal in vivo recombination, indicating that these residues are needed for a function that is not directly related to DNA binding or catalysis.

Amino Acid Sequence↗
PubMed1997

DNA binding of Escherichia coli arginine repressor mutants altered in oligomeric state.

The Escherichia coli arginine repressor (ArgR) controls expression of the arginine biosynthetic genes and acts as an accessory protein in Xer site-specific recombination at cer and related plasmid recombination sites. The hexameric wild-type protein shows L-arginine-dependent DNA binding. In this work, ArgR mutants that are defective in trimer-trimer interactions and bind DNA as trimers in an L-arginine-independent manner are isolated and characterized. Whereas the wild-type ArgR hexamer exhibits high-affinity binding to two repeated ARG boxes separated by 3 bp (each ARG box containing two identical dyad symmetrical 9 bp half-sites), the trimeric mutants bind to and footprint three adjacent half-sites of this 'idealized' substrate. Trimeric ArgR is impaired in its ability to repress the arginine biosynthetic genes and in Xer site-specific recombination. In the absence of L-arginine, residual wild-type ArgR-binding occurs as trimers. The binding of an N-terminal 77-amino-acid DNA-binding domain to idealized ARG boxes is also characterized.

Arginine↗
PubMed1996

Xer-mediated site-specific recombination in vitro.

The Xer site-specific recombination system acts at ColE1 cer and pSC101 psi sites to ensure that these plasmids are in a monomeric state prior to cell division. We show that four proteins, ArgR, PepA, XerC and XerD are necessary and sufficient for recombination between directly repeated cer sites on a supercoiled plasmid in vitro. Only PepA, XerC and XerD are required for recombination at psi in vitro. Recombination at cer and psi in vitro requires negative supercoiling and is exclusively intramolecular. Strand exchange at cer produces Holliday junction-containing products in which only the top strands have been exchanged. This reaction requires the catalytic tyrosine residue of Xer C but not that of XerD. Recombination at psi gives catenated circular resolution products. Strand exchange at psi is sequential. XerC catalyses the first (top) strand exchange to make a Holiday junction intermediate and XerD catalyses the second (bottom) strand exchange.

Base Sequence↗
PubMed1995

Cooperative binding of Tn3 resolvase monomers to a functionally asymmetric binding site.

BACKGROUND: The inverted repeat is a common feature of protein-binding sites in DNA. The two-fold symmetry of the inverted repeat corresponds to the two-fold symmetry of the protein that binds to it. In most natural inverted-repeat binding sites, however, the DNA sequence does not have perfect two-fold symmetry. Our study of how a site-specific recombinase recognizes an inverted-repeat binding site indicates that such sequence asymmetry can be functionally important. RESULTS: Tn3 resolvase forms two complexes with the 34 base-pair binding site II of its recombination region, res. A resolvase monomer first binds at the left end of the site; a second monomer then binds cooperatively at the right end. In both complexes, the DNA is bent by resolvase. In contrast, the closely related gamma delta resolvase binds to site II mainly as a dimer. Insertion of 5 or 10 base pairs at the centre of the site does not prevent cooperative binding of two Tn3 resolvase subunits. The fully occupied site II has a very asymmetric structure. Reversal of the orientation of site II in res blocks recombination; thus, its asymmetric properties are functionally important. We propose a structure for the two-subunit complex formed with site II, based on our results and by analogy with the co-crystal structure of gamma delta resolvase bound to res site I. CONCLUSIONS: Deviations from perfect inverted-repeat symmetry in a resolvase-binding site lead to ordered binding of subunits, structural asymmetry of resolvase-DNA complexes, and asymmetric function.

Base Sequence↗
PubMed1995

Xer site-specific recombination in vitro.

Two related recombinases, XerC and XerD, belonging to the lambda integrase family of enzymes, are required for Xer site-specific recombination in vivo. In order to understand the roles of these proteins in the overall reaction mechanism, an in vitro recombination system using a synthetic Holliday junction-containing substrate has been developed. Recombination of this substrate is efficient and requires both XerC and XerD. However, only exchange of one pair of strands, the one corresponding to the conversion of the Holliday junction intermediate back to the substrate, has been observed. Recombination reactions using XerC and XerD derivatives that are mutant in their presumptive catalytic residues, or are maltose-binding fusion recombinase derivatives, have demonstrated that this pair of strand exchanges is catalysed by XerC. The site of XerC-mediated cleavage has been located to between the last nucleotide of the XerC binding site and the first nucleotide of the central region. Cleavage at this site generates a free 5'-OH and a covalent complex between XerC and the 3' end of the DNA.

Base Sequence↗
PubMed1995

Site-specific recombination in the replication terminus region of Escherichia coli: functional replacement of dif.

The replication terminus region of the Escherichia coli chromosome encodes a locus, dif, that is required for normal chromosome segregation at cell division. dif is a substrate for site-specific recombination catalysed by the related chromosomally encoded recombinases XerC and XerD. It has been proposed that this recombination converts chromosome multimers formed by homologous recombination back to monomers in order that they can be segregated prior to cell division. Strains mutant in dif, xerC or xerD share a characteristic phenotype, containing a variable fraction of filamentous cells with aberrantly positioned and sized nucleoids. We show that the only DNA sequences required for wild-type dif function in the terminus region of the chromosome are contained within 33 bp known to bind XerC and XerD and that putative active site residues of the Xer recombinases are required for normal chromosome segregation. We have also shown that recombination by the loxP/Cre system of bacteriophage P1 will suppress the phenotype of a dif deletion strain when loxP is inserted in the terminus region. Suppression of the dif deletion phenotype did not occur when either dif/Xer or loxP/Cre recombination acted at other positions in the chromosome close to oriC or within lacZ, indicating that site-specific recombination must occur within the replication terminus region in order to allow normal chromosome segregation.

Base Sequence↗
PubMed1995

The pCLIP plasmids: versatile cloning vectors based on the bacteriophage lambda origin of replication.

A series of general-purpose plasmid vectors based on the phage lambda origin of replication (ori) has been constructed. Each vector consists of a backbone plasmid encoding chloramphenicol resistance (CmR) and containing a unique HaeII site into which the lacZ alpha-complementing multiple cloning site (MCS) region of an established vector was inserted. To increase the cloning potential of the inserted MCS, superfluous restriction sites in the backbone were removed by a variety of techniques. The vectors, designated pCLIP (for CmR lambda ori integration proficient) plasmids, are of medium copy number and are compatible with most other vectors in common use. A total of 17 unique restriction sites in pCLIP8, pCLIP9, pCLIP18, pCLIP19 and pCLIP23 are available for cloning. As well as possessing the usual properties of vectors, the pCLIP plasmids are able to integrate reversibly into lambda prophage by homologous recombination. Thus, cloned DNA can be maintained in single or multiple copy at will. By integrating recombinant plasmids into appropriate deletion prophages followed by induction, phage::plasmid hybrids are produced which can be manipulated as phage. These properties are demonstrated using a test recombinant plasmid, pCLIPLEU2. The pCLIP vectors are therefore useful for routine plasmid cloning, complementation analysis and applications where the ability to manipulate recombinants in plasmid, phage or prophage forms is advantageous.

Amino Acid Sequence↗