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
Tn3 resolvase catalyses recombination between a pair of identical res sites, directly repeated on a supercoiled plasmid molecule. The normal in vitro product is a two-noded catenane. A plasmid with a single base substitution at one res site is converted to non-recombinant knots by resolvase; the first major product is a four-noded knot. The linkage change, delta Lk, on formation of the four-noded knot is +4. The formation of the observed knots from the mutant substrate, and this value of delta Lk were predicted by and in agreement with a "simple rotation" model for catalysis of strand exchange by resolvase. A number of alternative mechanisms for knotting by resolvase are inconsistent with the observed topological changes.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Modelos Genéticos , Conformação de Ácido Nucleico , Nucleotidiltransferases/metabolismo , Recombinação Genética/genética , DNA Super-Helicoidal/metabolismo , Eletroforese em Gel de Ágar , Eletroforese em Gel Bidimensional/métodos , Plasmídeos/metabolismo , TransposasesRESUMO
In order to investigate the functions of the parts of the Tn 3 recombination site res, we created hybrid recombination sites by placing the loxP site for Cre recombinase adjacent to the "accessory" resolvase-binding sites II and III of res. The efficiency and product topology of in vitro recombination by Cre between two of these hybrid sites were affected by the addition of Tn 3 resolvase. The effects of resolvase addition were dependent on the relative orientation and spacing of the elements of the hybrid sites. Substrates with sites II and III of res close to loxP gave specific catenated or knotted products (four-noded catenane, three-noded knot) when resolvase and Cre were added together. The product topological complexity increased when the length of the spacer DNA segment between loxP and res site II was increased. Similar resolvase-induced effects on Cre recombination product topology were observed in reactions of substrates with loxP sites adjacent to full res sites. The results demonstrate that the res accessory sites are sufficient to impose topological selectivity on recombination, and imply that intertwining of two sets of accessory sites defines the simple catenane product topology in normal resolvase-mediated recombination. They are also consistent with current models for the mechanism of catalysis by Cre.
Assuntos
Bacteriófago P1/enzimologia , Integrases/metabolismo , Recombinação Genética , Transposases/metabolismo , Proteínas Virais , Sítios de Ligação , Técnicas Genéticas , Recombinases , Sequências Repetitivas de Ácido Nucleico , Especificidade por SubstratoRESUMO
Resolvases and DNA invertases catalyse site-specific recombination by a concerted cut-and-religate mechanism. Topological data strongly suggest a rotational movement of the DNA half-sites during recombination: in an "iterative" mode of reaction, after cleavage of all four strands of the two recombining sites, the recombinase-linked half-sites seem to rotate through multiple steps of 180 degrees prior to final religation. However, current structural data provide no clear support for the postulated corresponding rotation of enzyme subunits within an active tetramer. A key issue is whether repetition of apparent 180 degrees rotation steps requires rejoining of the DNA strands and resetting of the catalytic machinery, or if multiple rotation steps can take place in the fully cleaved intermediate. We present evidence that a resolvase-catalysed DNA knotting reaction, brought about by apparent 360 degrees rotation, can proceed without rejoining of the DNA strands in the recombinant (180 degrees rotation) configuration. This behaviour is not compatible with a mechanism requiring a fixed arrangement of the catalytic subunits, and strongly suggests that recombination is coupled to disruption of the dimer interface between two subunits bound at each crossover site. We also show that an artificial supercoiled plasmid containing two res sites, with a single mismatched base-pair in one of the crossover sites, is a substrate for "suicidal" reactions in which resolvase remains covalently linked to two half-sites.
Assuntos
DNA Nucleotidiltransferases/metabolismo , DNA/química , DNA/metabolismo , Recombinação Genética , Composição de Bases , Sequência de Bases , Sítios de Ligação , Catálise , Enzimas de Restrição do DNA , Proteínas de Ligação a DNA/metabolismo , Cinética , Modelos Estruturais , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Plasmídeos/metabolismo , Especificidade por Substrato , TransposasesRESUMO
Site-specific recombination catalysed by Tn3 resolvase requires the formation of an intermediate synaptic complex containing two res recombination sites and several resolvase subunits. Synaptic complexes were observed directly by chemical crosslinking of resolvase subunits followed by agarose gel electrophoresis. The highest yield of synaptic complex was from a "standard" substrate, a supercoiled plasmid with res sites in direct repeat, but complexes were also made between sites in inverted repeat, or in nicked or linear molecules, or in separate molecules. The substrate selectivity for synapsis is less stringent than for recombination; thus recombination selectivity is dependent on steps after synapsis. The stability of the synapse after its formation might be a key factor, since unproductive synapses are less stable than productive ones. In a standard substrate, synapsis is fast relative to the rate of recombination. Crosslinking in active reaction mixtures yields synaptic complexes derived from both the substrate and the catenane recombination product. Although catalysis of strand exchange is at binding site I of res, a pair of isolated site 1's do not synapse, whereas a synaptic complex is formed from a plasmid carrying two copies of res binding sites II and III. Our data are consistent with a model in which the formation of the synaptic intermediate is driven, and its structure defined, by the initial interaction of these accessory sites.
Assuntos
DNA Nucleotidiltransferases/metabolismo , DNA Circular/genética , Proteínas de Ligação a DNA/metabolismo , Recombinação Genética/genética , Reagentes de Ligações Cruzadas , DNA/genética , DNA/metabolismo , DNA Circular/metabolismo , Glutaral , Plasmídeos/genética , Plasmídeos/metabolismo , TransposasesRESUMO
Site-specific recombination, transposition, and retroviral integration reactions involve the collaborative action of multiple identical protein subunits, making it difficult to determine the catalytic functions and fate of a subunit at any particular DNA binding site of the substrate. To investigate the mechanism of catalysis by the site-specific recombinase Tn3 resolvase, we fixed specific subunits to their binding sites by laser photocrosslinking, using a partially synthetic supercoiled DNA substrate containing photoreactive nucleotides. Crosslinked resolvase subunits were able to participate in a complete recombination reaction, demonstrating that the interaction of the subunit with its binding site persists throughout the reaction, and thus placing limitations on acceptable models for the catalytic mechanism.
Assuntos
DNA Nucleotidiltransferases/metabolismo , DNA Super-Helicoidal/metabolismo , Recombinação Genética , Sequência de Bases , Sítios de Ligação , Reagentes de Ligações Cruzadas , Lasers , Dados de Sequência Molecular , Especificidade por Substrato , TransposasesAssuntos
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 ViralAssuntos
Antibacterianos/biossíntese , Genética Microbiana , Streptomyces/metabolismo , Ágar , Amino Açúcares/biossíntese , Antibacterianos/análise , Bioensaio , Metabolismo dos Carboidratos , Precipitação Química , Cromatografia em Papel , Cromatografia em Camada Fina , Meios de Cultura , Densitometria , Eletrodos , Fermentação , Filtração , Liofilização , Variação Genética , Glicosídeos/biossíntese , Concentração de Íons de Hidrogênio , Infecções por Klebsiella/metabolismo , Pigmentos Biológicos/biossíntese , Especificidade da Espécie , Esporos Bacterianos/crescimento & desenvolvimento , Streptomyces/crescimento & desenvolvimento , Streptomyces/efeitos da radiação , Raios UltravioletaRESUMO
Site-specific recombination catalyzed by Tn3 resolvase proceeds with a linkage change, delta Lk, of +4 in the forward resolution reaction and -4 in the catenane fusion reverse reaction. The reverse reaction occurs only at low superhelical densities and gives unknotted circular products, consistent with plectonemic and not solenoidal wrapping of the two recombination sites. The strand exchange topologies are consistent with a mechanism in which resolvase cleaves all four DNA strands and religates them after a 180 degrees rotation of two duplex partners in a right-handed sense for the "forward" reaction, and in a left-handed sense for the "reverse" action. This could be achieved by a 180 degrees rotation of two resolvase subunits within a tetramer with D2 symmetry; we suggest that a different symmetry applies to phage lamda integrase catalysis.
Assuntos
Elementos de DNA Transponíveis , DNA Super-Helicoidal/ultraestrutura , Nucleotidiltransferases/metabolismo , Recombinação Genética , Cloroquina , Eletroforese em Gel de Ágar/métodos , Conformação de Ácido Nucleico , Plasmídeos , TransposasesRESUMO
The resolvase-catalysed reaction between two res sites in a circular DNA substrate normally gives two circular recombination products linked in a two-noded catenane. Homology between the two res sites at the central overlap dinucleotide of subsite I is important for recombination. Reactions between res sites differing at one position in the central dinucleotide (AC X AT) gave a low yield of recombinants containing mismatched base-pairs, but gave large amounts of a non-recombinant four-noded knot. This result was predicted by a 'simple rotation' model for strand exchange. The mismatch is evidently recognized only after commitment to an initial 180 degrees rotation of the resolvase-linked DNA ends, and it induces a second 180 degrees rotation which restores correct base-pairing at the overlap, giving the four-noded product. Correct base-pairing is not essential for religation, but may be important for release of the products. Characteristic patterns of 4, 6, 8 and 10 node knots, or 4, 8, 12 and 16 node knots were obtained, depending on the reaction conditions and the resolvase. Two pathways for multiple rounds of rotation in 360 degrees steps are inferred. The results support a model for strand exchange by supercoil-directed subunit rotation within a resolvase tetramer.
Assuntos
DNA/metabolismo , Nucleotidiltransferases/metabolismo , Sequência de Bases , Catálise , DNA/genética , Eletroforese em Gel de Ágar , Glicosídeo Hidrolases/metabolismo , Cinética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Plasmídeos , Recombinação Genética , Especificidade por Substrato , Transposases , beta-FrutofuranosidaseRESUMO
Communications between distant sites on DNA often depend on the way in which the sites are connected. For example, site-specific recombination catalysed by Tn3 resolvase is most efficient when the 114-base-pair res recombination sites are directly repeated in the same DNA molecule. In vitro a supercoiled plasmid substrate containing two directly repeated res sites gives a resolution product in which the two recombinant circles are topologically linked as a simple (two-noded) catenane (Fig. 1a). Resolvase is highly selective in forming this product rather than unlinked circles or more complex catenanes. It does not catalyse recombination between sites on separate supercoiled molecules, or between inverted sites in the same supercoiled molecule. Tn3 resolution removes four negative supercoils from the substrate, an energetically favourable change which may drive the reaction: in relaxed or nicked circular substrates, resolution is incomplete and slower. Resolvase can catalyse fusion of the circles of a nicked or relaxed catenane, giving a single unknotted circular product. The fusion is the precise topological reversal of resolution, introducing four negative supercoils into a relaxed catenane substrate, and should therefore not proceed if the catenane is already negatively supercoiled. Here we study recombination between res sites in non-supercoiled DNA circles linked into simple catenanes. We used (+2) and (-2) catenanes, which differ only in the direction in which one circle is threaded through the other (Fig. 2a). Although stereoselectivity is a feature of enzyme catalysis, it is not obvious how resolvase can distinguish between these subtly different catenane diastereomers. A model for the intertwining of the res site DNA in the catalytically active complex predicts that only the (-2) catenane will recombine, giving unknotted and 4-noded knot circular products. We have confirmed this prediction for the Tn3 and Tn21 resolvases.
Assuntos
DNA Circular/metabolismo , DNA Super-Helicoidal/metabolismo , Nucleotidiltransferases/metabolismo , Recombinação Genética , Transposon Resolvases , DNA Circular/química , DNA Circular/genética , DNA Super-Helicoidal/química , DNA Super-Helicoidal/genética , Conformação Molecular , Conformação de Ácido Nucleico , Plasmídeos , TransposasesRESUMO
The Staphylococcus aureus transposon Tn552 encodes a protein (p480) containing the 'D,D(35)E' motif common to retroviral integrases and the transposases of a number of bacterial elements, including phage Mu, the integron-containing element Tn5090, Tn7 and IS3. p480 and a histidine-tagged derivative were overexpressed in Escherichia coli and purified by methods involving denaturation and renaturation. DNase I footprinting and gel binding assays demonstrated that p480 binds to two adjacent, directly repeated 23 bp motifs at each end of Tn552. Although donor strand cleavage by p480 was not detected, in vitro conditions were defined for strand transfer activity with transposon end fragments having pre-cleaved 3' termini. Strand transfer was Mn(2+)-dependent and appeared to join a single left or right end fragment to target DNA. The importance of the terminal dinucleotide CA-3' was demonstrated by mutation. The in vitro activities of p480 are consistent with its proposed function as the Tn552 transposase.
Assuntos
Elementos de DNA Transponíveis/genética , DNA Bacteriano/metabolismo , Nucleotidiltransferases/metabolismo , Staphylococcus aureus/enzimologia , Sequência de Bases , Proteínas de Transporte , Escherichia coli/genética , Rearranjo Gênico , Modelos Genéticos , Dados de Sequência Molecular , Nucleotidiltransferases/genética , Nucleotidiltransferases/isolamento & purificação , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Recombinação Genética , Especificidade por Substrato , TransposasesRESUMO
The serine recombinase gamma delta resolvase performs site-specific recombination in an elaborate synaptic complex containing 12 resolvase subunits and two 114-base pair res sites. Here we present an alternative structural model for the synaptic complex. Resolvase subunits in the complex contact their neighbors in equivalent ways, using three principal interactions, one of which is a newly proposed synaptic interaction. Evidence in support of this interaction is provided by mutations at the interface that either enable resolvase to synapse two copies of site I or inhibit synapsis of complete res sites. In our model, the two crossover sites are far apart, separated by the resolvase catalytic domains bound to them. Thus, recombination would require a substantial rearrangement of resolvase subunits or domains.
Assuntos
DNA/metabolismo , Modelos Moleculares , Recombinação Genética/fisiologia , Transposases/química , Transposases/metabolismo , Transposon Resolvases , Substâncias Macromoleculares , Conformação de Ácido Nucleico , Conformação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Subunidades Proteicas , Recombinases , Transposases/genéticaRESUMO
Tn3 resolvase promotes site-specific recombination between two res sites, each of which has three resolvase dimer-binding sites. Catalysis of DNA-strand cleavage and rejoining occurs at binding site I, but binding sites II and III are required for recombination. We used an in vivo screen to detect resolvase mutants that were active on res sites with binding sites II and III deleted (that is, only site I remaining). Mutations of amino acids Asp102 (D102) or Met103 (M103) were sufficient to permit catalysis of recombination between site I and a full res, but not between two copies of site I. A double mutant resolvase, with a D102Y mutation and an additional activating mutation at Glu124 (E124Q), recombined substrates containing only two copies of site I, in vivo and in vitro. In these novel site Ixsite I reactions, product topology is no longer restricted to the normal simple catenane, indicating synapsis by random collision. Furthermore, the mutants have lost the normal specificity for directly repeated sites and supercoiled substrates; that is, they promote recombination between pairs of res sites in linear molecules, or in inverted repeat in a supercoiled molecule, or in separate molecules.