RESUMO
Site-specific DNA recombinases play a variety of biological roles, often related to the dissemination of antibiotic resistance, and are also useful synthetic biology tools. The simplest site-specific recombination systems will recombine any two cognate sites regardless of context. Other systems have evolved elaborate mechanisms, often sensing DNA topology, to ensure that only one of multiple possible recombination products is produced. The closely related resolvases from the Tn3 and γδ transposons have historically served as paradigms for the regulation of recombinase activity by DNA topology. However, despite many proposals, models of the multi-subunit protein-DNA complex (termed the synaptosome) that enforces this regulation have been unsatisfying due to a lack of experimental constraints and incomplete concordance with experimental data. Here, we present new structural and biochemical data that lead to a new, detailed model of the Tn3 synaptosome, and discuss how it harnesses DNA topology to regulate the enzymatic activity of the recombinase.
Site-specific DNA recombinases alter the connectivity of DNA by recognizing specific DNA sequences, then cutting the DNA strands and pasting them together in a new configuration. Such enzymes play a variety of biological roles, often related to the dissemination of antibiotic resistance, and are also useful biotechnology tools. The simplest site-specific recombination systems will recombine any two cognate sites regardless of context. However, others have evolved elaborate mechanisms to ensure that only one of multiple possible recombination products is produced. Tn3 resolvase has long been known to be regulated by DNA topologythat is, it will cut and reconnect two target sequences only if they lie on the same DNA molecule, and if they are in the proper relative orientation. This study presents new structural and biochemical data that lead to a new, detailed model of the large proteinDNA complex formed by Tn3 resolvase and its cognate sites. This 3D model illustrates how DNA topology can be harnessed to regulate the activity of a recombinase and provides a basis for engineering Tn3 resolvase and related recombination systems as genome editing tools.
Assuntos
DNA , Complexos Multiproteicos , Transposon Resolvases , Elementos de DNA Transponíveis , Recombinases/genética , Transposases/genética , Transposon Resolvases/genética , Transposon Resolvases/metabolismo , Complexos Multiproteicos/químicaRESUMO
The site-specific recombinase Tn3 resolvase initiates DNA strand exchange when two res recombination sites and six resolvase dimers interact to form a synapse. The detailed architecture of this intricate recombination machine remains unclear. We have clarified which of the potential dimer-dimer interactions are required for synapsis and recombination, using a novel complementation strategy that exploits a previously uncharacterized resolvase from Bartonella bacilliformis ("Bart"). Tn3 and Bart resolvases recognize different DNA motifs, via diverged C-terminal domains (CTDs). They also differ substantially at N-terminal domain (NTD) surfaces involved in dimerization and synapse assembly. We designed NTD-CTD hybrid proteins, and hybrid res sites containing both Tn3 and Bart dimer binding sites. Using these components in in vivo assays, we demonstrate that productive synapsis requires a specific "R" interface involving resolvase NTDs at all three dimer-binding sites in res. Synapses containing mixtures of wild-type Tn3 and Bart resolvase NTD dimers are recombination-defective, but activity can be restored by replacing patches of Tn3 resolvase R interface residues with Bart residues, or vice versa. We conclude that the Tn3/Bart family synapse is assembled exclusively by R interactions between resolvase dimers, except for the one special dimer-dimer interaction required for catalysis.
Assuntos
Proteínas de Bactérias/metabolismo , Bartonella bacilliformis/metabolismo , Transposon Resolvases/metabolismo , Proteínas de Bactérias/genética , Bartonella bacilliformis/genética , Sítios de Ligação , DNA Nucleotidiltransferases/metabolismo , Elementos de DNA Transponíveis , Proteínas de Ligação a DNA/metabolismo , Dimerização , Domínios e Motivos de Interação entre Proteínas , Estrutura Quaternária de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Transposon Resolvases/genéticaRESUMO
An essential feature of many site-specific recombination systems is their ability to regulate the direction and topology of recombination. Resolvases from the serine recombinase family assemble an interwound synaptic complex that harnesses negative supercoiling to drive the forward reaction and promote recombination between properly oriented sites. To better understand the interplay of catalytic and regulatory functions within these synaptic complexes, we have solved the structure of the regulatory site synapse in the Sin resolvase system. It reveals an unexpected synaptic interface between helix-turn-helix DNA-binding domains that is also highlighted in a screen for synapsis mutants. The tetramer defined by this interface provides the foundation for a robust model of the synaptic complex, assembled entirely from available crystal structures, that gives insight into how the catalytic activity of Sin and other serine recombinases may be regulated.
Assuntos
Proteínas de Bactérias/química , DNA Nucleotidiltransferases/química , DNA/química , Modelos Moleculares , Recombinação Genética , Proteínas de Bactérias/genética , Sítios de Ligação , Catálise , Cristalização , Cristalografia por Raios X , DNA Nucleotidiltransferases/genética , Dimerização , Mutação , Conformação ProteicaRESUMO
Site-specific recombinases are enzymes that promote precise rearrangements of DNA sequences. They do this by cutting and rejoining the DNA strands at specific positions within a pair of target sites recognized and bound by the recombinase. One group of these enzymes, the serine recombinases, initiates strand exchange by making double-strand breaks in the DNA of the two sites, in an intermediate built around a catalytic tetramer of recombinase subunits. However, these catalytic steps are only the culmination of a complex pathway that begins when recombinase subunits recognize and bind to their target sites as dimers. To form the tetramer-containing reaction intermediate, two dimer-bound sites are brought together by protein dimer-dimer interactions. During or after this initial synapsis step, the recombinase subunit and tetramer conformations change dramatically by repositioning of component subdomains, bringing about a transformation of the enzyme from an inactive to an active configuration. In natural serine recombinase systems, these steps are subject to elaborate regulatory mechanisms in order to ensure that cleavage and rejoining of DNA strands only happen when and where they should, but we and others have identified recombinase mutants that have lost dependence on this regulation, thus facilitating the study of the basic steps leading to catalysis. We describe how our studies on activated mutants of two serine recombinases, Tn3 resolvase and Sin, are providing us with insights into the structural changes that occur before catalysis of strand exchange, and how these steps in the reaction pathway are regulated.
Assuntos
DNA Nucleotidiltransferases/metabolismo , DNA Nucleotidiltransferases/fisiologia , Recombinação Genética/fisiologia , Animais , Pareamento Cromossômico/genética , Pareamento Cromossômico/fisiologia , DNA Nucleotidiltransferases/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/fisiologia , Larva/genética , Larva/metabolismo , Modelos Biológicos , Modelos Moleculares , Proteínas Mutantes/metabolismo , Proteínas Mutantes/fisiologia , Ligação Proteica/fisiologia , Recombinação Genética/genética , Serina/metabolismoRESUMO
The resolvase Sin regulates DNA strand exchange by assembling an elaborate interwound synaptosome containing catalytic and regulatory Sin tetramers, and an architectural DNA-bending protein. The crystal structure of the regulatory tetramer was recently solved, providing new insights into the structural basis for regulation. Here we describe the selection and characterization of two classes of Sin mutations that, respectively, bypass or disrupt the functions of the regulatory tetramer. Activating mutations, which allow the catalytic tetramer to assemble and function independently at site I (the crossover site), were found at approximately 20% of residues in the N-terminal domain. The most strongly activating mutation (Q115R) stabilized a catalytically active synaptic tetramer in vitro. The positions of these mutations suggest that they act by destabilizing the conformation of the ground-state site I-bound dimers, or by stabilizing the altered conformation of the active catalytic tetramer. Mutations that block activation by the regulatory tetramer mapped to just two residues, F52 and R54, supporting a functional role for a previously reported crystallographic dimer-dimer interface. We suggest how F52/R54 contacts between regulatory and catalytic subunits might promote assembly of the active catalytic tetramer within the synaptosome.
Assuntos
Proteínas de Bactérias/genética , DNA Nucleotidiltransferases/genética , Modelos Moleculares , Staphylococcus aureus/genética , Domínio Catalítico , Mutagênese , Mutação , Estrutura Quaternária de Proteína , Staphylococcus aureus/enzimologiaRESUMO
A remarkable feature of the serine resolvases is their regulation: the wild-type enzymes will catalyse intra- but not inter-molecular recombination, can sense the relative orientation of their sites and can exchange strands directionally, despite the fact that there is no net release of chemical bond energy. The key to this regulation is that they are only active within a large intertwined complex called the 'synaptosome'. Because substrate topology greatly facilitates (or, in other cases, inhibits) formation of the synaptosome, it acts as a 'topological filter'. Within the defined topology of the synaptosome, strand exchange releases supercoiling tension, providing an energy source to bias the reaction direction. The regulatory portion of this complex contains additional copies of the recombinase and sometimes other DNA-bending proteins. We are using a combination of X-ray crystallography, biochemistry and genetics to model the full synaptic complex and to understand how the regulatory portion activates the crossover-site-bound recombinases.
Assuntos
Recombinases/fisiologia , Serina/metabolismo , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , DNA/química , DNA/metabolismo , DNA Nucleotidiltransferases/química , DNA Nucleotidiltransferases/metabolismo , Ativação Enzimática/fisiologia , Humanos , Modelos Biológicos , Modelos Moleculares , Conformação de Ácido Nucleico , Conformação Proteica , Recombinases/química , Recombinases/metabolismo , Recombinação Genética/genética , Recombinação Genética/fisiologiaRESUMO
Sin resolvase is a site-specific serine recombinase that is normally controlled by a complex regulatory mechanism. A single mutation, Q115R, allows the enzyme to bypass the entire regulatory apparatus, such that no accessory proteins or DNA sites are required. Here, we present a 1.86 Å crystal structure of the Sin Q115R catalytic domain, in a tetrameric arrangement stabilized by an interaction between Arg115 residues on neighboring subunits. The subunits have undergone significant conformational changes from the inactive dimeric state previously reported. The structure provides a new high-resolution view of a serine recombinase active site that is apparently fully assembled, suggesting roles for the conserved active site residues. The structure also suggests how the dimer-tetramer transition is coupled to assembly of the active site. The tetramer is captured in a different rotational substate than that seen in previous hyperactive serine recombinase structures, and unbroken crossover site DNA can be readily modeled into its active sites.
Assuntos
Proteínas de Bactérias/química , DNA Nucleotidiltransferases/química , Staphylococcus aureus/enzimologia , Proteínas de Bactérias/genética , Domínio Catalítico , Simulação por Computador , Cristalografia por Raios X , DNA Nucleotidiltransferases/genética , Ativação Enzimática , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Mutação de Sentido Incorreto , Conformação de Ácido Nucleico , Oligonucleotídeos/química , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Secundária de ProteínaRESUMO
Serine recombinases promote specific DNA rearrangements by a cut-and-paste mechanism that involves cleavage of all four DNA strands at two sites recognized by the enzyme. Dissecting the order and timing of these cleavage events and the steps leading up to them is difficult because the cleavage reaction is readily reversible. Here, we describe assays using activated Sin mutants and a DNA substrate with a 3'-bridging phosphorothiolate modification that renders Sin-mediated DNA cleavage irreversible. We find that activating Sin mutations promote DNA cleavage rather than simply stabilize the cleavage product. Cleavage events at the scissile phosphates on complementary strands of the duplex are tightly coupled, and the overall DNA cleavage rate is strongly dependent on Sin concentration. When combined with analytical ultracentrifugation data, these results suggest that Sin catalytic activity and oligomerization state are tightly linked, and that activating mutations promote formation of a cleavage-competent oligomeric state that is normally formed only transiently within the full synaptic complex.
Assuntos
Proteínas de Bactérias/metabolismo , DNA Nucleotidiltransferases/metabolismo , DNA/metabolismo , Multimerização Proteica , Recombinação Genética , Proteínas de Bactérias/genética , DNA/síntese química , DNA Nucleotidiltransferases/genética , Modelos Moleculares , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Oligonucleotídeos/síntese química , Oligonucleotídeos/metabolismo , Estrutura Quaternária de Proteína , UltracentrifugaçãoRESUMO
The serine recombinase Sin requires a non-specific DNA-bending protein such as Hbsu for activity at its recombination site resH. Hbsu, and Sin subunits bound at site II of resH, together regulate recombination, ensuring selectivity for directly repeated resH sites by specifying assembly of an intertwined synapse. To investigate the role of the DNA-bending protein in defining the architecture of the synapse, we constructed a chimaeric recombination site (resF) which allows Hbsu to be substituted by IHF, binding specifically between site I (the crossover site) and site II. Two Sin dimers and one IHF dimer can bind together to the closely adjoining sites in resF, forming folded complexes. The precise position of the IHF site within the site I-site II spacer determines the conformation of these complexes, and also the reactivity of the resF sites in recombination assays. The data suggest that a sharp bend with a specific geometry is required in the spacer DNA, to bring the Sin dimers at sites I and II together in the correct relative orientation for synapse assembly and regulation, consistent with our model for a highly condensed synapse in which Hbsu/IHF has a purely architectural function.
Assuntos
Proteínas de Bactérias/química , DNA Nucleotidiltransferases/química , DNA Bacteriano/química , Proteínas de Ligação a DNA/química , Fatores Hospedeiros de Integração/química , Recombinação Genética , Sítios de Ligação Microbiológicos/genética , Bacteriófago lambda/genética , Sítios de Ligação , DNA Super-Helicoidal/química , Modelos Moleculares , Conformação ProteicaRESUMO
Sin recombinase from Staphylococcus aureus acts selectively on directly repeated resH sites, assembling an intertwined synapse in which exactly three supercoils are trapped between the points of strand exchange. Resolution requires the two Sin binding sites in resH (site I, where strand exchange occurs, and site II) and a non-specific DNA-bending protein (e.g. Hbsu). We show that a single amino acid substitution in Sin (I100T) is sufficient to relax the normal requirements for site II and Hbsu. Using this hyperactive protein, and the variant recombination site resH(AT), we investigate the roles of site II and Hbsu in synapsis and strand exchange. We conclude that Sin bound at site II, and Hbsu, act together to control site I alignment and the topology of the synapse, and to stimulate strand exchange.
Assuntos
Proteínas de Bactérias/fisiologia , DNA Nucleotidiltransferases/metabolismo , Proteínas de Ligação a DNA/fisiologia , Recombinação Genética , Staphylococcus aureus/enzimologia , Sítios de Ligação , DNA Nucleotidiltransferases/química , DNA Nucleotidiltransferases/genética , DNA Bacteriano/química , DNA Circular/química , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismoRESUMO
The Sin recombinase from Staphylococcus aureus builds a distinctive DNA-protein synaptic complex to regulate strand exchange. Sin binds at two sites within an 86 basepair (bp) recombination site, resH. We propose that inverted motifs at the crossover site, and tandem motifs at the regulatory site, are recognized by structurally disparate Sin dimers. An essential architectural protein, Hbsu, binds at a discrete central site in resH. Positions of Hbsu-induced DNA deformation coincide with natural targets for Tn552 integration. Remarkably, Sin has the same topological selectivity as Tn3 and gammadelta resolvases. Our model for the recombination synapse has at its core an assembly of four Sin dimers; Hbsu plays an architectural role that is taken by two resolvase dimers in models of the Tn3/gammadelta synapse.
Assuntos
Proteínas de Bactérias/fisiologia , DNA Nucleotidiltransferases/fisiologia , Elementos de DNA Transponíveis/fisiologia , DNA Bacteriano/metabolismo , DNA Super-Helicoidal/metabolismo , Recombinação Genética , Staphylococcus aureus/metabolismo , Proteínas de Bactérias/química , Sequência de Bases , Sítios de Ligação , DNA Nucleotidiltransferases/química , DNA Bacteriano/genética , DNA Super-Helicoidal/genética , Proteínas de Ligação a DNA/metabolismo , Dimerização , Modelos Genéticos , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Ligação Proteica , Recombinases , Sequências Reguladoras de Ácido Nucleico , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Staphylococcus aureus/genética , Staphylococcus aureus/ultraestrutura , Especificidade por Substrato , Transposases/química , Transposases/metabolismo , Transposon ResolvasesRESUMO
Catalysis of DNA recombination by Tn3 resolvase is conditional on prior formation of a synapse, comprising 12 resolvase subunits and two recombination sites (res). Each res binds a resolvase dimer at site I, where strand exchange takes place, and additional dimers at two adjacent 'accessory' binding sites II and III. 'Hyperactive' resolvase mutants, that catalyse strand exchange at site I without accessory sites, were selected in E. coli. Some single mutants can resolve a res x site I plasmid (that is, with one res and one site I), but two or more activating mutations are necessary for efficient resolution of a site I x site I plasmid. Site I x site I resolution by hyperactive mutants can be further stimulated by mutations at the crystallographic 2-3' interface that abolish activity of wild-type resolvase. Activating mutations may allow regulatory mechanisms of the wild-type system to be bypassed, by stabilizing or destabilizing interfaces within and between subunits in the synapse. The positions and characteristics of the mutations support a mechanism for strand exchange by serine recombinases in which the DNA is on the outside of a recombinase tetramer, and the tertiary/quaternary structure of the tetramer is reconfigured.