RESUMO
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.
Assuntos
DNA Nucleotidiltransferases/metabolismo , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/metabolismo , Sequência de Bases , Sítios de Ligação , Elementos de DNA Transponíveis , Escherichia coli , Dados de Sequência Molecular , Mutação , Plasmídeos , TransposasesRESUMO
Site-specific recombination reactions bring about controlled rearrangements of DNA molecules by cutting the DNA at precise points and rejoining the ends to new partners. The recombinases that catalyse these reactions can be grouped into two families by amino acid sequence homology. We describe our current understanding of how these proteins catalyse recombination, and show how the catalytic mechanisms of the two families differ.
Assuntos
DNA Nucleotidiltransferases/metabolismo , DNA/metabolismo , Rearranjo Gênico , Recombinação Genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Catálise , DNA/genética , DNA Nucleotidiltransferases/classificação , DNA Nucleotidiltransferases/genética , Elementos de DNA Transponíveis , DNA Circular/metabolismo , Integrases , Dados de Sequência Molecular , Família Multigênica , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Ligação Proteica , Recombinases , Homologia de Sequência do Ácido Nucleico , Especificidade por Substrato , Transposases , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Site-specific recombination processes in microbes bring about precise DNA rearrangements which have diverse and important biological functions. The sites and recombinase enzymes used for these processes fall into two distinct families. Here we describe how experiments with one family, exemplified by the resolution system of transposon Tn3, have provided insight into the ways in which DNA and protein interact to bring together distant recombination sites and promote strand exchange.
Assuntos
Nucleotidiltransferases/fisiologia , Recombinação Genética , Troca Genética , Elementos de DNA Transponíveis , TransposasesRESUMO
The ability to visualise specific genes and proteins within bacterial cells is revolutionising knowledge of chromosome segregation. The essential elements appear to be the driving force behind DNA replication, which occurs at fixed cellular positions, the condensation of newly replicated DNA by a chromosome condensation machine located at the cell 1/4 and 3/4 positions, and molecular machines that act at midcell to allow chromosome separation after replication and movement of the sister chromosomes away from the division septum prior to cell division. This review attempts to provide a perspective on current views of the bacterial chromosome segregation mechanism and how it relates to other cellular processes.
Assuntos
Bactérias/genética , Segregação de Cromossomos , Cromossomos Bacterianos/genética , Compartimento Celular , Divisão Celular , Replicação do DNARESUMO
We report the construction of two novel Escherichia coli strains (DH1lacdapD and DH1lacP2dapD) that facilitate the antibiotic-free selection and stable maintenance of recombinant plasmids in complex media. They contain the essential chromosomal gene, dapD, under the control of the lac operator/promoter. Unless supplemented with IPTG (which induces expression of dapD) or DAP, these cells lyse. However, when the strains are transformed with a multicopy plasmid containing the lac operator, the operator competitively titrates the LacI repressor and allows expression of dapD from the lac promoter. Thus transformants can be isolated and propagated simply by their ability to grow on any medium by repressor titration selection. No antibiotic resistance genes or other protein expressing sequences are required on the plasmid, and antibiotics are not necessary for plasmid selection, making these strains a valuable tool for therapeutic DNA and recombinant protein production. We describe the construction of these strains and demonstrate plasmid selection and maintenance by repressor titration, using the new pORT plasmid vectors designed to facilitate recombinant DNA exploitation.
Assuntos
Cromossomos Bacterianos/genética , Resistência Microbiana a Medicamentos/genética , Escherichia coli/genética , Plasmídeos/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Escherichia coli/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica , Marcadores Genéticos , Canamicina/farmacologia , Óperon Lac/genética , Dados de Sequência Molecular , Transformação GenéticaRESUMO
We report here modifications of human beta-globin PAC clones by homologous recombination in Escherichia coli DH10B, utilising a plasmid temperature sensitive for replication, the recA gene and a wild-type copy of the rpsL gene which allows for an efficient selection for plasmid loss in this host. High frequencies of recombination are observed even with very small lengths of homology and the method has general utility for introducing insertions, deletions and point mutations. No rearrangements were detected with the exception of one highly repetitive genomic sequence when either the E.COLI: RecA- or the lambdoid phage encoded RecT and RecE-dependent recombination systems were used.
Assuntos
Clonagem Molecular/métodos , Globinas/genética , Bacteriófago P1/genética , Escherichia coli , Proteínas de Escherichia coli , Genes Bacterianos , Marcadores Genéticos , Vetores Genéticos , Globinas/metabolismo , Humanos , Plasmídeos , Recombinases Rec A/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Recombinação Genética , Proteína S9 RibossômicaRESUMO
In bacteria, two categories of specialised recombination promote a variety of DNA rearrangements. Transposition is the process by which genetic elements move between different locations of the genome, whereas site-specific recombination is a reaction in which DNA strands are broken and exchanged at precise positions of two target DNA loci to achieve determined biological function. Both types of recombination are represented by diverse genetic systems which generally encode their own recombination enzymes. These enzymes, generically called transposases and site-specific recombinases, can be grouped into several families on the basis of amino acid sequence similarities, which, in some cases, are limited to a signature of a few residues involved in catalysis. The well characterised site-specific recombinases are found to belong to two distinct groups whereas the transposases form a large super-family of enzymes encompassing recombinases from both prokaryotes and eukaryotes. In spite of important differences in the catalytic mechanisms used by these three classes of enzymes to cut and rejoin DNA molecules, similar strategies are used to coordinate the biochemical steps of the recombination reaction and to control its outcome. This review summarises our current understanding of transposition and site-specific recombination, attempting to illustrate how relatively conserved DNA cut-and-paste mechanisms can be used to bring about a variety of complex DNA rearrangements.
Assuntos
DNA Bacteriano/genética , Integrases , Recombinação Genética , Bactérias/genética , DNA Nucleotidiltransferases/metabolismo , Elementos de DNA Transponíveis , DNA Bacteriano/metabolismo , Rearranjo Gênico , Glicosídeo Hidrolases/metabolismo , Modelos Genéticos , Nucleoproteínas/genética , Nucleoproteínas/metabolismo , Recombinases , Transposases/metabolismo , Transposon Resolvases , beta-FrutofuranosidaseRESUMO
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.
Assuntos
DNA Nucleotidiltransferases/genética , DNA Bacteriano/genética , Proteínas de Escherichia coli , Escherichia coli/genética , Integrases , Recombinação Genética , Sequência de Bases , DNA Nucleotidiltransferases/metabolismo , DNA Bacteriano/metabolismo , Dados de Sequência Molecular , Plasmídeos , Ligação Proteica/genética , Recombinases , Análise de Sequência de DNARESUMO
Xer site-specific recombination at the psi site from plasmid pSC101 displays topological selectivity, such that recombination normally occurs only between directly repeated sites on the same circular DNA molecule. This intramolecular selectivity is important for the biological role of psi, and is imposed by accessory proteins PepA and ArcA acting at accessory DNA sequences adjacent to the core recombination site. Here we show that the selectivity for intramolecular recombination at psi can be bypassed in multiply interlinked catenanes. Xer site-specific recombination occurred relatively efficiently between antiparallel psi sites located on separate rings of right-handed torus catenanes containing six or more nodes. This recombination introduced one additional node into the catenanes. Antiparallel sites on four-noded right-handed catenanes, the normal product of Xer recombination at psi, were not recombined efficiently. Furthermore, parallel psi sites on right-handed torus catenanes were not substrates for Xer recombination. These findings support a model in which psi sites are plectonemically interwrapped, trapping a precise number of supercoils that are converted to four catenation nodes by Xer strand exchange.
Assuntos
Bacteriófago lambda/enzimologia , DNA Nucleotidiltransferases/metabolismo , DNA/metabolismo , Integrases/metabolismo , Recombinação Genética , DNA/química , Conformação de Ácido Nucleico , Oligodesoxirribonucleotídeos , Plasmídeos , Recombinases , Especificidade por SubstratoRESUMO
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.
Assuntos
DNA Nucleotidiltransferases/metabolismo , Proteínas de Escherichia coli , Integrases , Alquilação , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Divisão Celular , DNA Nucleotidiltransferases/química , DNA Nucleotidiltransferases/genética , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Escherichia coli/citologia , Escherichia coli/enzimologia , Escherichia coli/genética , Mutação , Recombinases , Recombinação Genética , Especificidade por SubstratoRESUMO
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.
Assuntos
Proteínas de Bactérias/metabolismo , Bacteriófago T7/enzimologia , DNA Bacteriano/metabolismo , Desoxirribonuclease I/metabolismo , Endodesoxirribonucleases/metabolismo , Proteínas de Escherichia coli , Escherichia coli/enzimologia , Conformação de Ácido Nucleico , Recombinação Genética , DNA Bacteriano/química , DNA Super-Helicoidal , PlasmídeosRESUMO
Site-specific recombinases XerC and XerD function in the segregation of circular bacterial replicons. In a recombining nucleoprotein complex containing two molecules each of XerC and XerD, coordinated reciprocal switches in recombinase activity ensure that only XerC or XerD is active at any one time. Mutated recombinases that carry sub?stitutions of a catalytic arginine residue stimulate cleavage and strand exchange mediated by the partner recombinase on DNA substrates that are normally recombined poorly by the partner. This is associated with a reciprocal impairment of the recombinase's own ability to initiate catalysis. The extent of this switch in catalysis is modulated by changes in recombination site sequence and is not a direct consequence of any catalytic defect. We propose that altered interactions between the mutated proteins and their wild-type partners lead to an increased level of an alternative Holliday junction intermediate that has a conformation appropriate for resolution by the partner recombinase. The results indicate how subtle changes in protein-DNA architecture at a Holliday junction can redirect recombination outcome.
Assuntos
DNA Nucleotidiltransferases/metabolismo , Proteínas de Escherichia coli , Escherichia coli/enzimologia , Escherichia coli/genética , Integrases , Recombinação Genética/genética , Substituição de Aminoácidos/genética , Arginina/genética , Arginina/metabolismo , Sequência de Bases , Sítios de Ligação , Catálise , DNA Nucleotidiltransferases/antagonistas & inibidores , DNA Nucleotidiltransferases/química , DNA Nucleotidiltransferases/genética , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Ativação Enzimática , Cinética , Mutação/genética , Conformação de Ácido Nucleico , Fenótipo , Ligação Proteica , Recombinases , Sequências Reguladoras de Ácido Nucleico/genética , Especificidade por SubstratoRESUMO
Xer-mediated site-specific recombination contributes to the stability of circular chromosomes in bacteria by resolving plasmid multimers and chromosome dimers to monomers prior to cell division. Two related site-specific recombinases, XerC and XerD, each catalyse one pair of strand exchange during Xer recombination. In order to relate the recently determined structure of XerD to its function, the XerD protein was subjected to pentapeptide scanning mutagenesis, which leads to a variable five amino acid cassette being introduced randomly into the target protein. This has allowed identification of regions of XerD involved in specific DNA binding, in communicating with the partner recombinase, XerC, and in catalysis and its control. The C-terminal domain of XerD, comprising two-thirds of the protein, contains the catalytic active site and comprises ten alpha helices (alphaE to alphaN) and a beta hairpin. A flexible linker connects this domain to the N-terminal domain that comprises four alpha helices (alphaA to alphaD). Pentapeptide insertions into alphaB, alphaD, alphaG, or alphaJ interfered with DNA binding. Helices alphaG and alphaJ comprise a pseudo helix-turn-helix DNA binding motif that may provide specificity of recombinase binding. An insertion in alphaL, adjacent to an active site arginine residue, led to loss of cooperative interactions between XerC and XerD and abolished recombination activity. Other insertions close to active site residues also abolished recombination activity. Proteins with an insertion in the beta hairpin turn bound DNA, interacted cooperatively with XerC and had a phenotype that is consistent with the protein being defective in XerD catalysis. This beta hairpin appears to be highly conserved in related proteins. Insertions at a number of dispersed locations did not impair XerD catalytic activity or DNA binding, but failed to allow XerC catalysis in vivo, indicating that several sites of interaction between XerD and XerC may be important for activation of XerC catalysis by XerD.
Assuntos
DNA Nucleotidiltransferases/química , DNA Nucleotidiltransferases/genética , Integrases , Mutagênese Insercional , Oligopeptídeos/química , Oligopeptídeos/genética , Sequência de Aminoácidos , Catálise , Sequência Conservada , DNA Nucleotidiltransferases/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Immunoblotting , Modelos Moleculares , Dados de Sequência Molecular , Oligopeptídeos/metabolismo , Ligação Proteica/genética , Recombinases , Recombinação Genética , Salmonella typhimurium/enzimologia , Salmonella typhimurium/genética , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Bacteriófago lambda/genética , Vetores Genéticos , Plasmídeos , Origem de Replicação , Sequência de Aminoácidos , Sequência de Bases , Resistência ao Cloranfenicol/genética , Clonagem Molecular , DNA Recombinante , Escherichia coli/genética , Fator F , Genes Reporter , Vetores Genéticos/genética , Dados de Sequência Molecular , Plasmídeos/genética , Recombinação GenéticaRESUMO
The Xer site-specific recombination system of Escherichia coli resolves both chromosome dimers and multimers of certain plasmids including those of ColE1. In this manner, Xer site-specific recombination contributes to the accurate distribution of circular chromosomes at cell division. Two related site-specific recombinases, XerC and XerD, are required for this process. The xerC and xerD genes of Salmonella typhimurium LT2 were isolated from libraries of LT2 genomic DNA by genetic complementation of E. coli Xer mutants. The putative proteins specified by the S. typhimurium genes can substitute for and are highly homologous to the corresponding proteins in E. coli. The distribution of amino acid dissimilarities differs, however, between pairs of cognate Xer proteins. The immediate genetic contexts of equivalent xer genes, i.e., in operons with genes of apparently unrelated function, are conserved between the two bacteria. This is the first description of the identification of a pair of functional homologues of the xerC and xerD genes of E. coli.
Assuntos
Cromossomos Bacterianos/fisiologia , DNA Nucleotidiltransferases/genética , Proteínas de Escherichia coli , Integrases , Salmonella typhimurium/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Teste de Complementação Genética , Dados de Sequência Molecular , Óperon , Filogenia , Recombinases , Recombinação Genética , Mapeamento por Restrição , Alinhamento de Sequência , Homologia de Sequência de AminoácidosAssuntos
Bacteriófago mu/genética , Elementos de DNA Transponíveis/genética , Elementos Facilitadores Genéticos , Regulação Viral da Expressão Gênica , Bacteriófago mu/fisiologia , Elementos de DNA Transponíveis/fisiologia , Modelos Genéticos , Recombinação Genética , Seleção Genética , Replicação ViralRESUMO
Plasmid ColE1 specifies a recombination site (cer) which participates in the conversion of plasmid dimers to monomers. The uncontrolled accumulation of dimers (and higher oligomeric forms) would otherwise lead to plasmid instability. Exonuclease III-generated deletions have been used to define the left-hand boundary of the cer site. Deletions which have lost up to 60 bp adjacent to the boundary no longer mediate the conversion of plasmid dimers to monomers, but still recombine with a wild-type site. Although this boundary region is essential for dimer resolution, its DNA sequence is poorly conserved among multimer resolution sites in related plasmids. We present evidence that its function is to influence the three-dimensional organization of the site and suggest that it may be required for the formation of a condensed nucleoprotein complex.
Assuntos
DNA Bacteriano/genética , Plasmídeos , Recombinação Genética , Deleção Cromossômica , DNA Bacteriano/metabolismo , Escherichia coli , Conformação Molecular , Homologia de Sequência do Ácido NucleicoRESUMO
Mobilization of the plasmid ColE1 from cells containing a conjugative plasmid (such as F) requires the synthesis of ColE1 mob proteins, and the presence, in cis, of bom (basis of mobility), a region of ColE1 containing the origin of transfer (oriT). The process of ColE1 transfer is thought to resemble that of the conjugative plasmid F, although the plasmids share little sequence homology. In F, conjugation is preceded by a strand-specific nicking event at oriT. The nicked strand is then conducted to the recipient with the 5' end leading. This is believed also to occur with ColE1, but direct biochemical confirmation has been precluded by its small size (6.65 kb). To test this hypothesis genetically, a novel method, using a lambda dv-based vector, has been devised to site-specifically integrate bom (or any other cloned sequence) into the chromosome of Escherichia coli. When provided with suitable mobilizing plasmids, such strains were found to transfer the chromosome in a polar way. From these data, the orientation of transfer of ColE1 was deduced and shown to be analogous to F.
Assuntos
Plasmídeos de Bacteriocinas , Cromossomos Bacterianos/fisiologia , Escherichia coli/genética , Plasmídeos , Clonagem Molecular , Enzimas de Restrição do DNA , Vetores Genéticos , Genótipo , Homologia de Sequência do Ácido NucleicoRESUMO
The Xer site-specific recombination system of Escherichia coli is involved in the stable inheritance of circular replicons. Multimeric replicons, produced by homologous recombination, are converted to monomers by the action of two related recombinases XerC and XerD. Site-specific recombination at a locus, dif, within the chromosomal replication terminus region is thought to convert dimeric chromosomes to monomers, which can then be segregated prior to cell division. The recombinases XerC and XerD bind cooperatively to dif, where they catalyse recombination. Chemical modification of specific bases and the phosphate-sugar backbone within dif was used to investigate the requirements for binding of the recombinases. Site-directed mutagenesis was then used to alter bases implicated in recombinase binding. Characterization of these mutants by in vitro recombinase binding and in vivo recombination, has demonstrated that the cooperative interactions between XerC and XerD can partially overcome DNA alterations that should interfere with specific recombinase-dif interactions.