RESUMO
The site-specific recombination pathway of bacteriophage λ encompasses isoenergetic but highly directional and tightly regulated integrative and excisive reactions that integrate and excise the vial chromosome into and out of the bacterial chromosome. The reactions require 240 bp of phage DNA and 21 bp of bacterial DNA comprising 16 protein binding sites that are differentially used in each pathway by the phage-encoded Int and Xis proteins and the host-encoded integration host factor and factor for inversion stimulation proteins. Structures of higher-order protein-DNA complexes of the four-way Holliday junction recombination intermediates provided clarifying insights into the mechanisms, directionality, and regulation of these two pathways, which are tightly linked to the physiology of the bacterial host cell. Here we review our current understanding of the mechanisms responsible for regulating and executing λ site-specific recombination, with an emphasis on key studies completed over the last decade.
Assuntos
Bacteriófago lambda , Recombinação Genética , Bacteriófago lambda/genética , Bacteriófago lambda/fisiologia , DNA Viral/genética , DNA Viral/metabolismo , Proteínas Virais/metabolismo , Proteínas Virais/genética , DNA Bacteriano/metabolismo , DNA Bacteriano/genética , Sítios de Ligação , Fatores Hospedeiros de Integração/metabolismo , Fatores Hospedeiros de Integração/genéticaRESUMO
The molecular machinery responsible for DNA expression, recombination, and compaction has been difficult to visualize as functionally complete entities due to their combinatorial and structural complexity. We report here the structure of the intact functional assembly responsible for regulating and executing a site-specific DNA recombination reaction. The assembly is a 240-bp Holliday junction (HJ) bound specifically by 11 protein subunits. This higher-order complex is a key intermediate in the tightly regulated pathway for the excision of bacteriophage λ viral DNA out of the E. coli host chromosome, an extensively studied paradigmatic model system for the regulated rearrangement of DNA. Our results provide a structural basis for pre-existing data describing the excisive and integrative recombination pathways, and they help explain their regulation.
Assuntos
Bacteriófago lambda/genética , DNA Bacteriano/química , DNA Cruciforme/química , DNA Viral/química , Proteínas de Escherichia coli/química , Escherichia coli/genética , Recombinação Genética , Microscopia Crioeletrônica , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , DNA Viral/genética , DNA Viral/metabolismo , Imageamento Tridimensional , Modelos MolecularesRESUMO
The site-specific recombinase encoded by bacteriophage λ (Int) is responsible for integrating and excising the viral chromosome into and out of the chromosome of its Escherichia coli host. Int carries out a reaction that is highly directional, tightly regulated, and depends upon an ensemble of accessory DNA bending proteins acting on 240 bp of DNA encoding 16 protein binding sites. This additional complexity enables two pathways, integrative and excisive recombination, whose opposite, and effectively irreversible, directions are dictated by different physiological and environmental signals. Int recombinase is a heterobivalent DNA binding protein and each of the four Int protomers, within a multiprotein 400 kDa recombinogenic complex, is thought to bind and, with the aid of DNA bending proteins, bridge one arm- and one core-type DNA site. In the 12 years since the publication of the last review focused solely on the λ site-specific recombination pathway in Mobile DNA II, there has been a great deal of progress in elucidating the molecular details of this pathway. The most dramatic advances in our understanding of the reaction have been in the area of X-ray crystallography where protein-DNA structures have now been determined for of all of the DNA-protein interfaces driving the Int pathway. Building on this foundation of structures, it has been possible to derive models for the assembly of components that determine the regulatory apparatus in the P-arm, and for the overall architectures that define excisive and integrative recombinogenic complexes. The most fundamental additional mechanistic insights derive from the application of hexapeptide inhibitors and single molecule kinetics.
Assuntos
Bacteriófago lambda/enzimologia , Integrases/metabolismo , Recombinação Genética , Cristalografia por Raios X , DNA/metabolismo , Escherichia coli/virologia , Integrases/química , Modelos Biológicos , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , Conformação ProteicaRESUMO
The virally encoded site-specific recombinase Int collaborates with its accessory DNA bending proteins IHF, Xis, and Fis to assemble two distinct, very large, nucleoprotein complexes that carry out either integrative or excisive recombination along regulated and essentially unidirectional pathways. The core of each complex consists of a tetramer of Integrase protein (Int), which is a heterobivalent DNA binding protein that binds and bridges a core-type DNA site (where strand cleavage and ligation are executed), and a distal arm-type site, that is brought within range by one or more DNA bending proteins. The recent determination of the patterns of these Int bridges has made it possible to think realistically about the global architecture of the recombinogenic complexes. Here, we combined the previously determined Int bridging patterns with in-gel FRET experiments and in silico modeling to characterize and differentiate the two 400-kDa multiprotein Holiday junction recombination intermediates formed during λ integration and excision. The results lead to architectural models that explain how integration and excision are regulated in λ site-specific recombination. Our confidence in the basic features of these architectures is based on the redundancy and self-consistency of the underlying data from two very different experimental approaches to establish bridging interactions, a set of strategic intracomplex distances from FRET experiments, and the model's ability to explain key aspects of the integrative and excisive recombination pathways, such as topological changes, the mechanism of capturing attB, and the features of asymmetry and flexibility within the complexes.
Assuntos
Bacteriófago lambda/genética , Bacteriófago lambda/fisiologia , DNA Cruciforme/genética , DNA Cruciforme/metabolismo , Lisogenia/genética , Nucleoproteínas/genética , Nucleoproteínas/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Ativação Viral/genética , Sítios de Ligação , Simulação por Computador , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , DNA Cruciforme/química , Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/virologia , Transferência Ressonante de Energia de Fluorescência , Integrases/química , Integrases/genética , Integrases/metabolismo , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Conformação de Ácido Nucleico , Nucleoproteínas/química , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Recombinação Genética , Proteínas Virais/químicaRESUMO
The site-specific recombinase encoded by bacteriophage λ [λ Integrase (Int)] is responsible for integrating and excising the viral chromosome into and out of the chromosome of its Escherichia coli host. In contrast to the other well-studied and highly exploited tyrosine recombinase family members, such as Cre and Flp, Int carries out a reaction that is highly directional, tightly regulated, and depends on an ensemble of accessory DNA bending proteins acting on 240 bp of DNA encoding 16 protein binding sites. This additional complexity enables two pathways, integrative and excisive recombination, whose opposite, and effectively irreversible, directions are dictated by different physiological and environmental signals. Int recombinase is a heterobivalent DNA binding protein that binds via its small amino-terminal domain to high affinity arm-type DNA sites and via its large, compound carboxyl-terminal domain to core-type DNA sites, where DNA cleavage and ligation are executed. Each of the four Int protomers, within a multiprotein 400-kDa recombinogenic complex, is thought to bind and, with the aid of DNA bending proteins, bridge one arm- and one core-type DNA site. Despite a wealth of genetic, biochemical, and functional information generated by many laboratories over the last 50 y, it has not been possible to decipher the patterns of Int bridges, an essential step in understanding the architectures responsible for regulated directionality of recombination. We used site-directed chemical cross-linking of Int in trapped Holliday junction recombination intermediates and recombination reactions with chimeric recombinases, to identify the unique and monogamous patterns of Int bridges for integrative and excisive recombination.
Assuntos
Bacteriófago lambda/genética , Bacteriófago lambda/fisiologia , DNA Cruciforme/genética , DNA Cruciforme/metabolismo , Integrases/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Sítios de Ligação , Reagentes de Ligações Cruzadas , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , DNA Cruciforme/química , Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/virologia , Integrases/química , Integrases/genética , Modelos Moleculares , Conformação de Ácido Nucleico , Nucleoproteínas/química , Nucleoproteínas/genética , Nucleoproteínas/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Recombinação Genética , Proteínas Virais/química , Ativação Viral/genética , Integração Viral/genéticaRESUMO
Supercoiled DNA is the relevant substrate for a large number of DNA transactions and has additionally been found to be a favorable form for delivering DNA and protein-DNA complexes to cells. We report here a facile method for stoichiometrically incorporating several different modifications at multiple, specific, and widely spaced sites in supercoiled DNA. The method is based upon generating an appropriately gapped circular DNA, starting from single-strand circular DNA from two phagemids with oppositely oriented origins of replication. The gapped circular DNA is annealed with labeled and unlabeled synthetic oligonucleotides to make a multiply nicked circle, which is covalently sealed and supercoiled. The method is efficient, robust and can be readily scaled up to produce large quantities of labeled supercoiled DNA for biochemical and structural studies. We have applied this method to generate dye-labeled supercoiled DNA with heteroduplex bubbles for a Förster resonance energy transfer (FRET) analysis of supercoiled Holliday junction intermediates in the λ integrative recombination reaction. We found that a higher-order structure revealed by FRET in the supercoiled Holliday junction intermediate is preserved in the linear recombination product. We suggest that in addition to studies on recombination complexes, these methods will be generally useful in other reactions and systems involving supercoiled DNA.
Assuntos
DNA Super-Helicoidal/química , Recombinação Genética , Sítios de Ligação Microbiológicos , DNA Cruciforme/metabolismo , DNA Super-Helicoidal/metabolismo , Fluoresceína , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes , Técnicas Genéticas , Origem de Replicação , RodaminasRESUMO
From bacterial viruses to humans, site-specific recombination and transposition are the major pathways for rearranging genomes on both long- and short-time scales. The site-specific pathways can be divided into 2 groups based on whether they are stochastic or regulated. Recombinases Cre and lambda Int are well-studied examples of each group, respectively. Both have been widely exploited as powerful and flexible tools for genetic engineering: Cre primarily in vivo and lambda Int primarily in vitro. Although Cre and Int use the same mechanism of DNA strand exchange, their respective reaction pathways are very different. Cre-mediated recombination is bidirectional, unregulated, does not require accessory proteins, and has a minimal symmetric DNA target. We show that when Cre is fused to the small N-terminal domain of Int, the resulting chimeric Cre recombines complex higher-order DNA targets comprising >200 bp encoding 16 protein-binding sites. This recombination requires the IHF protein, is unidirectional, and is regulated by the relative levels of the 3 accessory proteins, IHF, Xis, and Fis. In one direction, recombination depends on the Xis protein, and in the other direction it is inhibited by Xis. It is striking that regulated directionality and complexity can be conferred in a simple chimeric construction. We suggest that the relative ease of constructing a chimeric Cre with these properties may simulate the evolutionary interconversions responsible for the large variety of site-specific recombinases observed in Archaea, Eubacteria, and Eukarya.
Assuntos
Integrases/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Sequência de Bases , Sítios de Ligação , Colífagos/genética , Primers do DNA , Integrases/genética , Reação em Cadeia da Polimerase , Proteínas Recombinantes de Fusão/genética , Recombinação GenéticaRESUMO
The site-specific recombinase integrase encoded by bacteriophage lambda promotes integration and excision of the viral chromosome into and out of its Escherichia coli host chromosome through a Holliday junction recombination intermediate. This intermediate contains an integrase tetramer bound via its catalytic carboxyl-terminal domains to the four "core-type" sites of the Holliday junction DNA and via its amino-terminal domains to distal "arm-type" sites. The two classes of integrase binding sites are brought into close proximity by an ensemble of accessory proteins that bind and bend the intervening DNA. We have used a biotin interference assay that probes the requirement for major groove protein binding at specified DNA loci in conjunction with DNA protection, gel mobility shift, and genetic experiments to test several predictions of the models derived from the x-ray crystal structures of minimized and symmetrized surrogates of recombination intermediates lacking the accessory proteins and their cognate DNA targets. Our data do not support the predictions of "non-canonical" DNA targets for the N-domain of integrase, and they indicate that the complexes used for x-ray crystallography are more appropriate for modeling excisive rather than integrative recombination intermediates. We suggest that the difference in the asymmetric interaction profiles of the N-domains and arm-type sites in integrative versus excisive recombinogenic complexes reflects the regulation of recombination, whereas the asymmetry of these patterns within each reaction contributes to directionality.
Assuntos
Sítios de Ligação Microbiológicos , Bacteriófago lambda/enzimologia , Bioensaio/métodos , Biotina/metabolismo , Integrases/metabolismo , Recombinação Genética/genética , Sítios de Ligação , DNA Nucleotidiltransferases/metabolismo , DNA Cruciforme/química , Modelos Biológicos , Conformação de Ácido Nucleico , Ligação Proteica , Estrutura Terciária de Proteína , Deleção de SequênciaRESUMO
The highly directional and tightly regulated recombination reaction used to site-specifically excise the bacteriophage lambda chromosome out of its E. coli host chromosome requires the binding of six sequence-specific proteins to a 99 bp segment of the phage att site. To gain structural insights into this recombination pathway, we measured 27 FRET distances between eight points on the 99 bp regulatory DNA bound with all six proteins. Triangulation of these distances using a metric matrix distance-geometry algorithm provided coordinates for these eight points. The resulting path for the protein-bound regulatory DNA, which fits well with the genetics, biochemistry, and X-ray crystal structures describing the individual proteins and their interactions with DNA, provides a new structural perspective into the molecular mechanism and regulation of the recombination reaction and illustrates a design by which different families of higher-order complexes can be assembled from different numbers and combinations of the same few proteins.
Assuntos
Sítios de Ligação Microbiológicos/genética , DNA Bacteriano/química , Proteínas de Ligação a DNA/química , Integrases/química , Recombinação Genética , Algoritmos , Bacteriófago lambda , Sequência de Bases , Cristalografia por Raios X , DNA Nucleotidiltransferases/química , Proteínas de Escherichia coli/química , Fator Proteico para Inversão de Estimulação , Transferência Ressonante de Energia de Fluorescência , Integrases/fisiologia , Modelos Moleculares , Dados de Sequência Molecular , Fatores de Transcrição/química , Proteínas Virais/químicaRESUMO
The site-specific recombination pathway by which the bacteriophage lambda chromosome is excised from its Escherichia coli host chromosome is a tightly regulated, highly directional, multistep reaction that is executed by a series of multiprotein complexes. Until now, it has been difficult to study the individual steps of such reactions in the context of the entire pathway. Using single-molecule light microscopy, we have examined this process from start to finish. Stable bent-DNA complexes containing integrase and the accessory proteins IHF (integration host factor) and Xis form rapidly on attL and attR recombination partners, and synapsis of partner complexes follows rapidly after their formation. Integrase-mediated DNA cleavage before or immediately after synapsis is required to stabilize the synaptic assemblies. Those complexes that synapsed (approximately 50% of the total) yield recombinant product with a remarkable approximately 100% efficiency. The rate-limiting step of excision occurs after synapsis, but closely precedes or is concomitant with the appearance of a stable Holliday junction. Our kinetic analysis shows that directionality of this recombination reaction is conferred by the irreversibility of multiple reaction steps.
Assuntos
Sítios de Ligação Microbiológicos/genética , Bacteriófago lambda/genética , Recombinação Genética/genética , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , DNA Viral/genética , DNA Viral/metabolismo , Escherichia coli/genética , Escherichia coli/virologia , Integrases/metabolismo , Cinética , Mutação/genéticaRESUMO
The integrase protein of bacteriophage lambda (Int) catalyzes site-specific recombination between lambda phage and Escherichia coli genomes. Int is a tyrosine recombinase that binds to DNA core sites via a C-terminal catalytic domain and to a collection of arm DNA sites, distant from the site of recombination, via its N-terminal domain. The arm sites, in conjunction with accessory DNA-bending proteins, provide a means of regulating the efficiency and directionality of Int-catalyzed recombination. Recent crystal structures of lambda Int tetramers bound to synaptic and Holliday junction intermediates, together with new biochemical data, suggest a mechanism for the allosteric control of the recombination reaction through arm DNA binding interactions.
Assuntos
Bacteriófago lambda/genética , DNA Viral/fisiologia , Integrases/fisiologia , Recombinação Genética , Integração Viral/fisiologia , Bacteriófago lambda/enzimologia , Integrases/genéticaRESUMO
Bacteriophage lambda integrase (Int) catalyzes the integration and excision of the phage lambda chromosome into and out of the Esherichia coli host chromosome. The seven carboxy-terminal residues (C-terminal tail) of Int comprise a context-sensitive regulatory element that links catalytic function with protein multimerization and also coordinates Int functions within the multimeric recombinogenic complex. The experiments reported here show that the beta5-strand of Int is not simply a placeholder for the C-terminal tail but rather exerts its own allosteric effects on Int function in response to the incoming tail. Using a mutant integrase in which the C-terminal tail has been deleted (W350ter), we demonstrate that the C-terminal tail is required for efficient and accurate resolution of Holliday junctions by tetrameric Int. Addition of a free heptameric peptide of the same sequence as the C-terminal tail partially reverses the W350ter defects by stimulating Holliday junction resolution. The peptide also stimulates the topoisomerase function of monomeric W350ter. Single residue alterations in the peptide sequence and a mutant of the beta5 strand indicate that the observed stimulation arises from specific contacts with the beta5 strand (residues 239-243). The peptide does not stimulate binding of W350ter to its cognate DNA sites and therefore appears to recapitulate the effects of the normal C-terminal tail intermolecular contacts in wild-type Int. Models for the allosteric stimulation of Int activity by beta5 strand contacts are discussed.
Assuntos
Bacteriófago lambda/enzimologia , Integrases/química , Integrases/genética , Recombinação Genética , Sítio Alostérico , Bacteriófago lambda/genética , Cromossomos Bacterianos/metabolismo , Cristalografia por Raios X , DNA/química , DNA Cruciforme , Relação Dose-Resposta a Droga , Escherichia coli/metabolismo , Modelos Genéticos , Modelos Moleculares , Conformação Molecular , Conformação de Ácido Nucleico , Peptídeos/química , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas/química , Fatores de TempoRESUMO
Site-specific DNA recombination is important for basic cellular functions including viral integration, control of gene expression, production of genetic diversity and segregation of newly replicated chromosomes, and is used by bacteriophage lambda to integrate or excise its genome into and out of the host chromosome. lambda recombination is carried out by the bacteriophage-encoded integrase protein (lambda-int) together with accessory DNA sites and associated bending proteins that allow regulation in response to cell physiology. Here we report the crystal structures of lambda-int in higher-order complexes with substrates and regulatory DNAs representing different intermediates along the reaction pathway. The structures show how the simultaneous binding of two separate domains of lambda-int to DNA facilitates synapsis and can specify the order of DNA strand cleavage and exchange. An intertwined layer of amino-terminal domains bound to accessory (arm) DNAs shapes the recombination complex in a way that suggests how arm binding shifts the reaction equilibrium in favour of recombinant products.
Assuntos
Bacteriófago lambda/enzimologia , DNA Cruciforme/química , DNA Cruciforme/metabolismo , Integrases/química , Integrases/metabolismo , Recombinação Genética/genética , Regulação Alostérica , Sítios de Ligação Microbiológicos/genética , Sequência de Bases , Catálise , Cristalografia por Raios X , DNA Cruciforme/genética , Isomerismo , Modelos Moleculares , Conformação de Ácido Nucleico , Maleabilidade , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
Lambda integrase (Int) mediates recombination between attachment sites on phage and Escherichia coli DNA. Int is assisted by accessory protein-induced DNA loops in bridging pairs of distinct "arm-type" and "core-type" DNA sites to form synapsed recombination complexes that subsequently recombine by means of a Holliday junction (HJ) intermediate. An in-gel FRET assay was developed and used to measure 15 distances between six points in two Int-HJ complexes containing arm-DNA oligonucleotides, and 3D maps of these complexes were derived by distance-geometry calculations. The maps reveal unexpected positions for the arm-type DNAs relative to core sites on the HJ and a new Int conformation in the HJ tetramer. The results show how the position of arm DNAs determines the bias of catalytic activities responsible for directional resolution, provide insights into the organization of Int higher-order complexes, and lead to models of the structure of the full HJ recombination intermediates.
Assuntos
Bacteriófago lambda/enzimologia , DNA Cruciforme/química , Transferência Ressonante de Energia de Fluorescência , Integrases/química , DNA Cruciforme/metabolismo , Eletroforese em Gel de Poliacrilamida , Integrases/metabolismo , Fatores de TempoRESUMO
Lambda integrase (Int) forms higher-order protein-DNA complexes necessary for site-specific recombination. The carboxy-terminal domain of Int (75-356) is responsible for catalysis at specific core-type binding sites whereas the amino-terminal domain (1-70) is responsible for cooperative arm-type DNA binding. Alanine scanning mutagenesis of residues 64-70, within full-length integrase, has revealed differential effects on cooperative arm binding interactions that are required for integrative and excisive recombination. Interestingly, while these residues are required for cooperative arm-type binding on both P'1,2 and P'2,3 substrates, cooperative binding at the arm-type sites P'2,3 was more severely compromised than binding at arm-type sites P'1,2 for L64A. Concomitantly, L64A had a much stronger effect on integrative than on excisive recombination. The arm-binding properties of Int appear to be intrinsic to the amino-terminal domain because the phenotype of L64A was the same in an amino-terminal fragment (Int 1-75) as it was in the full-length protein.
Assuntos
Integrases/genética , Integrases/metabolismo , Sequência de Bases , Cromossomos Bacterianos/genética , Pegada de DNA , DNA Bacteriano/genética , Desoxirribonuclease I , Escherichia coli/enzimologia , Mutagênese , Deleção de SequênciaRESUMO
The bacteriophage lambda site-specific recombinase (Int), in contrast to other family members such as Cre and Flp, has an amino-terminal domain that binds "arm-type" DNA sequences different and distant from those involved in strand exchange. This defining feature of the heterobivalent recombinases confers a directionality and regulation that is unique among all recombination pathways. We show that the amino-terminal domain is not a simple "accessory" element, as originally thought, but rather is incorporated into the core of the recombination mechanism, where it is well positioned to exert its profound effects. The results reveal an unexpected pattern of intermolecular interactions between the amino-terminal domain of one protomer and the linker region of its neighbor within the tetrameric Int complex and provide insights into those features distinguishing an "active" from an "inactive" pair of Ints during Holliday junction resolution.
Assuntos
Bacteriófago lambda/enzimologia , DNA Cruciforme , Integrases/metabolismo , Alelos , Sequência de Bases , Primers do DNA , Integrases/químicaRESUMO
The integrase protein (Int) of phage lambda is a well-studied representative of the tyrosine recombinase family, whose defining features are two sequential pairs of DNA cleavage/ligation reactions that proceed via a 3' phosphotyrosine covalent intermediate to first form and then resolve a Holliday junction recombination intermediate. We devised an assay that takes advantage of DNA hairpin formation at one Int target site to trap Int cleavages at a different target site, and thereby reveal iterative cycles of cleavage and ligation that would otherwise be undetected. Using this assay and others to compare wild-type Int and a mutant (R169D) defective in forming proper dimer/tetramer interfaces, we found that the efficiency of "bottom-strand" DNA cleavage by wild-type Int, but not R169D, is very sensitive to the base-pair at the "top-strand" cleavage site, seven base-pairs away. We show that this is related to the finding that hairpin formation involving ligation of a mispaired base is much faster for R169D than for wild-type Int, but only in the context of a multimeric complex. During resolution of Holliday junction recombination intermediates, wild-type Int, but not R169D, is very sensitive to homology at the sites of ligation. A long-sought insight from these results is that during Holliday junction resolution the tetrameric Int complex remains intact until after ligation of the product helices has been completed. This contrasts with models in which the second pair of DNA cleavages is a trigger for dissolution of the recombination complex.
Assuntos
Bacteriófago lambda/genética , Reparo do DNA/genética , DNA Bacteriano/metabolismo , Integrases/química , Integrases/metabolismo , Recombinação Genética , Bacteriófago lambda/enzimologia , DNA Nucleotidiltransferases/genética , DNA Nucleotidiltransferases/metabolismo , DNA Bacteriano/química , Dimerização , Integrases/genética , Modelos Moleculares , Mutação , Conformação de Ácido NucleicoRESUMO
Despite many years of genetic and biochemical studies on the lambda integrase (Int) recombination system, it is still not known whether the Int protein is competent for DNA cleavage as a monomer. We have addressed this question, as part of a larger study of Int functions critical for the formation of higher-order complexes, by isolating "multimer-specific" mutants. We identify a pair of oppositely charged residues, E153 and R169, that comprise an intermolecular salt bridge within a functional Int multimer. Mutation of either of these residues significantly reduces both the cleavage of full-att sites and the resolution of Holliday junctions without compromising the cleavage of half-att site substrates. Allele-specific suppressor mutations were generated at these residues. Their interaction with wild-type Int on preformed Holliday junctions indicates that the mutated residues comprise an intermolecular salt bridge. We have also shown that the most C-terminal seven residues of Int, which comprise another previously identified subunit interface, inhibit DNA cleavage by monomeric but not multimeric Int. Taken together, our results lead us to conclude that Int can cleave DNA as a monomer. We also identify and discuss unique structural features of Int that act negatively to reduce its activity as a monomer and other features that act positively to enhance its activity as a multimer.
Assuntos
Bacteriófago lambda/enzimologia , Integrases/química , Integrases/metabolismo , Substituição de Aminoácidos , Sequência de Bases , Sítios de Ligação , Primers do DNA , DNA Cruciforme/química , DNA Cruciforme/metabolismo , Cinética , Substâncias Macromoleculares , Modelos Moleculares , Mutagênese Sítio-Dirigida , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
The bacteriophage lambda integrase protein (lambda Int) belongs to a family of tyrosine recombinases that catalyze DNA rearrangements. We have determined a crystal structure of lambda Int complexed with a cleaved DNA substrate through a covalent phosphotyrosine bond. In comparison to an earlier unliganded structure, we observe a drastic conformational change in DNA-bound lambda Int that brings Tyr342 into the active site for cleavage of the DNA in cis. A flexible linker connects the central and the catalytic domains, allowing the protein to encircle the DNA. Binding specificity is achieved through direct interactions with the DNA and indirect readout of the flexibility of the att site. The conformational switch that activates lambda Int for DNA cleavage exposes the C-terminal 8 residues for interactions with a neighboring Int molecule. The protein interactions mediated by lambda Int's C-terminal tail offer a mechanism for the allosteric control of cleavage activity in higher order lambda Int complexes.