DNA binding properties of TnpX indicate that different synapses are formed in the excision and integration of the Tn4451 family

Site-specific recombination is an important mechanism for genetic exchange. Insertional recombination mediated by the recently delineated large resolvase or serine recombinase proteins is unique within the resolvase family as integration was thought to be a reaction catalysed only by members of the integrase or tyrosine recombinase family of site-specific recombinases. The large resolvase TnpX is a serine recombinase that is responsible for the movement of the Tn4451/3 family of chloramphenicol resistance elements, which are found within two genera of the medically important clostridia. Deletion analysis of TnpX showed that the last 110 amino acids (aa) of TnpX, which comprise a cysteine rich region, were not essential for its biological function and that a region required for DNA binding was located between aa 493-597. Purified TnpX was shown to bind to the ends of the element and to the joint of the circular intermediate with high affinity but, most unusually, to bind to its target sites with a considerably lower affinity. Therefore, it was concluded that the resolvase-like excision and insertion reactions mediated by TnpX were distinct processes even though the same serine recombinase mechanism was involved. TnpX is the first large serine recombinase in which differential binding to its transposon and target sites has been demonstrated.     

Two distinct regions of the large serine recombinase TnpX are required for DNA binding and biological function

The large serine recombinase, TnpX, from the Clostridium perfringens integrative mobilizable element Tn 4451 , consists of three domains and has two known DNA binding regions. In this study random and site-directed mutagenesis was used to identify other regions of TnpX that were required for biological activity. Genetic and biochemical analysis of these mutants led to the identification of important TnpX residues in the N-terminal catalytic pocket. In addition, another region of TnpX (aa 243–261), which is conserved within large serine recombinases, was shown to be essential for both excision and insertion. Mutation of charged residues within this region led to a loss of biological activity and aberrant DNA binding. This phenotype was mediated by interaction with the distal DNA binding region (aa 598–707). In these mutants, removal of residues 598–707 resulted in loss of DNA binding, despite the presence of the primary DNA binding region (aa 533–583). Analysis of mutations within the aa 243–261 region indicated that different protein conformations were involved in the insertion and the excision reactions. In summary, we have shown that TnpX is a complex protein that has multiple intra- and intermolecular interaction sites, providing insight into the structural and functional complexity of this important enzyme family.     

Diversity in the serine recombinases

Most site-specific recombinases fall into one of two families, based on evolutionary and mechanistic relatedness. These are the tyrosine recombinases or lambda integrase family and the serine recombinases or resolvase/invertase family. The tyrosine recombinases are structurally diverse and functionally versatile and include integrases, resolvases, invertases and transposases. Recent studies have revealed that the serine recombinase family is equally versatile and members have a variety of structural forms. The archetypal resolvase/invertases are highly regulated, only affect resolution or inversion and they have an N-terminal catalytic domain and a C-terminal DNA binding domain. Phage-encoded serine recombinases (e.g. phiC31 integrase) cause integration and excision with strictly controlled directionality, and have an N-terminal catalytic domain but much longer C-terminal domains compared with the resolvase/invertases. This high molecular weight group also contains transposases (e.g. TnpX from Tn4451). Other transposases, which belong to a third structurally different group, are similar in size to the resolvase/invertases but have the DNA binding domain N-terminal to the catalytic domain (e.g. IS607 transposase). These three structural groups represented by the resolvase/invertases, the large serine recombinases and relatives of IS607 transposase correlate with three major groupings seen in a phylogeny of the catalytic domains. These observations indicate that the serine recombinases are modular and that fusion of the catalytic domain to unrelated sequences has generated structural and functional diversity.     

Solution structure and DNA binding of the catalytic domain of the large serine resolvase TnpX

The transfer of antibiotic resistance between bacteria is mediated by mobile genetic elements such as plasmids and transposons. TnpX is a member of the large serine recombinase subgroup of site‐specific recombinases and is responsible for the excision and insertion of mobile genetic elements that encode chloramphenicol resistance in the pathogens Clostridium perfringens and Clostridium difficile. TnpX consists of three structural domains: domain I contains the catalytic site, whereas domains II and III contain DNA‐binding motifs. We have solved the solution structure of residues 1–120 of the catalytic domain I of TnpX. The TnpX catalytic domain shares the same overall fold as other serine recombinases; however, differences are evident in the identity of the proposed hydrogen donor and in the size, amino acid composition, conformation, and dynamics of the TnpX active site loops. To obtain the interaction surface of TnpX_1–120, we titrated a DNA oligonucleotide containing the circular intermediate joint attCI recombination site into ¹⁵N‐labeled TnpX_1–120 and observed progressive nuclear magnetic resonance chemical shift perturbations using ¹⁵N HSQC spectra. Perturbations were largely confined to a region surrounding the catalytic serine and encompassed residues of the active site loops. Utilizing the perturbation map and the data‐driven docking program, HADDOCK, we have generated a model of the DNA interaction complex for the TnpX catalytic domain. Copyright © 2015 John Wiley & Sons, Ltd.     

The architecture of the gammadelta resolvase crossover site synaptic complex revealed by using constrained DNA substrates

Activated mutants of the serine recombinase, gammadelta resolvase, form a simplified recombinogenic synaptic complex containing a tetramer of resolvase and two crossover sites. We have probed the architecture of this complex by measuring the efficiency of recombination of a series of constrained DNA substrates (with phased recombination sites separated by an IHF-induced U-turn); this serves as a direct report on the topology of a productive synapse. Our data show that in the active complex, the catalytic domains from two resolvase dimers form a central core, while the DNA binding domains and the DNA lie on the outside. In addition, the crossover sites cross one another to form a local positive node. The implications of our data for the mechanism of strand exchange and the process of resolvase activation are discussed.     

Localization of the exonuclease and polymerase domains of Bacillus subtilis DNA polymerase III.

Structural gene mutants were cloned and exploited to identify the major catalytic domains of Bacillus subtilis DNA polymerase III (BsPolIII), a 162.4-kDa [1437 amino acids (aa)] polymerase: 3'-5' exonuclease (Exo) required for replicative DNA synthesis. Analysis of the sequence, mutagenicity, and catalytic behavior of natural and site-directed point mutants of BsPolIII unequivocally located the domain involved in exonuclease catalysis within a 155-aa residue segment displaying homology with the Exo domain of Escherichia coli DNA polymerase I. Sequence analysis of four structural gene mutations which specifically alter then enzyme's reactivity to the inhibitory dGTP analog, 6-(p-hydroxyphenylhydrazino)uracil, and the inhibitory arabinonucleotide, araCTP, defined a domain (Pol) involved in dNTP binding. The Pol domain was in the C-terminal fourth of the enzyme within a 98-aa segment spanning aa 1175-1273. The primary structure of the domain was unique, displaying no obvious conservation in any other DNA polymerase, including the distantly related PolIIIs of the Gram- organisms, E. coli and Salmonella typhimurium.     

The large resolvase TnpX is the only transposon-encoded protein required for transposition of the Tn4451/3 family of integrative mobilizable elements

Chloramphenicol resistance in Clostridium perfringens and Clostridium difficile is often encoded by catP genes located within the 6.3 kb integrative mobilizable elements Tn4451 and Tn4453 respectively. This family of transposons is capable of being mobilized into a recipient cell in the presence of another conjugative element. Transposition is mediated by the large resolvase TnpX, which excises the element to produce a circular molecule that is the integrative intermediate. In this study, in vivo deletion analysis of the transposon-encoded tnpV and tnpY genes showed that they are not essential for excision or integration of this group of elements. Similar studies on tnpW suggested either that this gene is not essential for these functions or that TnpW does not function when provided in trans. Development and use of an in vivo insertion assay showed that TnpX is the only transposon-encoded protein required for the integration reaction. Subsequently, a TnpXLEH6 protein was purified and shown to catalyse excision in vitro in the absence of any other protein and preferentially to excise a supercoiled DNA substrate. In summary, these studies have shown that TnpX is the only transposon protein required in vivo and in vitro for the excision process and that, like excision, integration also occurs by a serine recombinase-mediated site-specific recombination mechanism.     

A comprehensive approach to zinc-finger recombinase customization enables genomic targeting in human cells

Zinc-finger recombinases (ZFRs) represent a potentially powerful class of tools for targeted genetic engineering. These chimeric enzymes are composed of an activated catalytic domain derived from the resolvase/invertase family of serine recombinases and a custom-designed zinc-finger DNA-binding domain. The use of ZFRs, however, has been restricted by sequence requirements imposed by the recombinase catalytic domain. Here, we combine substrate specificity analysis and directed evolution to develop a diverse collection of Gin recombinase catalytic domains capable of recognizing an estimated 3.77 × 10⁷ unique DNA sequences. We show that ZFRs assembled from these engineered catalytic domains recombine user-defined DNA targets with high specificity, and that designed ZFRs integrate DNA into targeted endogenous loci in human cells. This study demonstrates the feasibility of generating customized ZFRs and the potential of ZFR technology for a diverse range of applications, including genome engineering, synthetic biology and gene therapy.     

The activity of the murine DNA methyltransferase Dnmt1 is controlled by interaction of the catalytic domain with the N-terminal part of the enzyme leading to an allosteric activation of the enzyme after binding to methylated DNA

The mammalian DNA methyltransferase Dnmt1 is responsible for the maintenance of the pattern of DNA methylation in vivo. It is a large multidomain enzyme comprising 1620 amino acid residues. We have purified and characterized individual domains of Dnmt1 (NLS-containing domain, NlsD, amino acid residues: 1-343; replication foci-directing domain, 350-609; Zn-binding domain (ZnD), 613-748; polybromo domain, 746-1110; and the catalytic domain (CatD), 1124-1620). CatD, ZnD and NlsD bind to DNA, demonstrating the existence of three independent DNA-binding sites in Dnmt1. CatD shows a preference for binding to hemimethylated CpG-sites; ZnD prefers methylated CpGs; and NlsD specifically binds to CpG-sites, but does not discriminate between unmethylated and methylated DNA. These results are not compatible with the suggestion that the target recognition domain of Dnmt1 resides in the N terminus of the enzyme. We show by protein-protein interaction assays that ZnD and CatD interact with each other. The isolated catalytic domain does not methylate DNA, neither alone nor in combination with other domains. Full-length Dnmt1 was purified from baculovirus-infected insect cells. Under the experimental conditions, Dnmt1 has a strong (50-fold) preference for hemimethylated DNA. Dnmt1 is stimulated to methylate unmodified CpG sites by the addition of fully methylated DNA. This effect is dependent on Zn, suggesting that binding of methylated DNA to ZnD triggers the allosteric activation of the catalytic center of Dnmt1. The allosteric activation model can explain kinetic data obtained by others. It suggests that Dnmt1 might be responsible for spreading of methylation, a process that is observed during aging and carcenogenesis but may be important for de novo methylation of DNA.     

The resolvase/invertase domain of the site-specific recombinase TnpX is functional and recognizes a target sequence that resembles the junction of the circular form of the Clostridium perfringens transposon Tn4451.

Tn4451 is a 6.3-kb chloramphenicol resistance transposon from Clostridium perfringens and is found on the conjugative plasmid pIP401. The element undergoes spontaneous excision from multicopy plasmids in Escherichia coli and C. perfringens and conjugative excision from pIP401 in C. perfringens. Tn4451 is excised as a circular molecule which is probably the transposition intermediate. Excision of Tn4451 is dependent upon the site-specific recombinase TnpX, which contains potential motifs associated with both the resolvase/invertase and integrase families of recombinases. Site-directed mutagenesis of conserved amino acid residues within these domains was used to show that the resolvase/invertase domain was essential for TnpX-mediated excision of Tn4451 from multicopy plasmids in E. coli. An analysis of Tn4451 target sites revealed that the transposition process showed target site specificity. The Tn4451 target sequence resembled the junction of the circular form, and insertion occurred at a GA dinucleotide. Tn4451 insertions were flanked by directly repeated GA dinucleotides, and there was also a GA at the junction of the circular form, where the left and right termini of Tn4451 were fused. We propose a model for Tn4451 excision and insertion in which the resolvase/invertase domain of TnpX introduces 2-bp staggered cuts at these GA dinucleotides. Analysis of Tn4451 derivatives with altered GA dinucleotides provided experimental evidence to support the model.     

Changing the enzymatic activity of T7 endonuclease by mutations at the beta-bridge site: alteration of substrate specificity profile and metal ion requirements by mutation distant from the catalytic domain

Phage-encoded resolvase T7 endonuclease I is a structure-specific endonuclease. The enzyme acts on a broad spectrum of substrates with a variety of DNA structures. The enzyme is a dimer with two separated catalytic domains connected by an elongated beta-sheet bridge. The activities of enzymes with mutations in the beta-bridge segment were studied. Mutations that did not affect catalytic domain folding and function but did alter the relative positions of these domains retained catalytic activity but with altered specificity and metal ion dependence. Our results suggest that the enzyme recognizes its substrates by DNA conformation exclusion and offer a simple explanation for the broad substrate specificity of phage resolvase.     

A novel DNA binding and nuclease activity in domain III of Mu transposase: evidence for a catalytic region involved in donor cleavage.

The Mu A protein is a 75 kDa transposase organized into three structural domains. By severing the C-terminal region (domain III) from the remainder of the protein, we unmasked a novel non-specific DNA binding and nuclease activity in this region. Deletion analysis localized both activities to a 26 amino acid stretch (aa 575-600) which remarkably remained active in DNA binding and cleavage. The two activities were shown to be tightly linked by site-directed mutagenesis. To study the importance of these activities in the transposition process, an intact mutant transposase lacking the DNA binding and nuclease activity of domain III was constructed and purified. The mutant transposase was indistinguishable from wild-type Mu A in binding affinity for both the Mu ends and the enhancer, and in strand transfer activity when the cleavage step was bypassed. In contrast, the mutant transposase displayed defects in both synapsis and donor cleavage. Our results strongly suggest that the 26 amino acid region in domain III carries catalytic residues required for donor DNA cleavage by Mu A protein. Furthermore, our data suggest that an active site for donor cleavage activity in the Mu tetramer is assembled from domain II (metal ion binding) in one A monomer and domain III (DNA cleavage) in a separate A monomer. This proposal for active site assembly is in agreement with the recently proposed domain sharing model by Yang et al. (Yang, J.Y., Kim, K., Jayaram, M. and Harshey, R.M. [1995] EMBO J., 14, 2374-2384).     

Tetrameric structure of a serine integrase catalytic domain

The serine integrases have recently emerged as powerful new chromosome engineering tools in various organisms and show promise for therapeutic use in human cells. The serine integrases are structurally and mechanistically unrelated to the bacteriophage lambda integrase but share a similar catalytic domain with the resolvase/invertase enzymes typified by the resolvase proteins from transposons Tn3 and gammadelta. Here we report the crystal structure and solution properties of the catalytic domain from bacteriophage TP901-1 integrase. The protein is a dimer in solution but crystallizes as a tetramer that is closely related in overall architecture to structures of activated gammadelta-resolvase mutants. The ability of the integrase tetramer to explain biochemical experiments performed in the resolvase and invertase systems suggests that the TP901 integrase tetramer represents a unique intermediate on the recombination pathway that is shared within the serine recombinase superfamily.     

Secondary and tertiary structural changes in gamma delta resolvase: comparison of the wild-type enzyme, the I110R mutant, and the C-terminal DNA binding domain in solution.

gamma delta Resolvase is a site-specific DNA recombinase (M(r) 20.5 kDa) in Escherichia coli that shares homology with a family of bacterial resolvases and invertases. We have characterized the secondary and tertiary structural behavior of the cloned DNA binding domain (DBD) and a dimerization defective mutant in solution. Low-salt conditions were found to destabilize the tertiary structure of the DBD dramatically, with concomitant changes in the secondary structure that were localized near the hinge regions between the helices. The molten tertiary fold appears to contribute significantly to productive DNA interactions and supports a mechanism of DNA-induced folding of the tertiary structure, a process that enables the DBD to adapt in conformation for each of the three imperfect palindromic sites. At high salt concentrations, the monomeric I110R resolvase shows a minimal perturbation to the three helices of the DBD structure and changes in the linker segment in comparison to the cloned DBD containing the linker. Comparative analysis of the NMR spectra suggest that the I110R mutant contains a folded catalytic core of approximately 60 residues and that the segment from residues 100 to 149 are devoid of regular structure in the I110R resolvase. No increase in the helicity of the linker region of I110R resolvase occurs on binding DNA. These results support a subunit rotation model of strand exchange that involves the partial unfolding of the catalytic domains.     

Mechanism of DnaB helicase of Escherichia coli: structural domains involved in ATP hydrolysis, DNA binding, and oligomerization.

We describe the delineation of three distinct structural domains of the DnaB helicase of Escherichia coli: domain alpha, amino acid residues (aa) 1-156; domain beta, aa 157-302; and domain gamma, aa 303-471. Using mutants with deletion in these domains, we have examined their role(s) in hexamer formation, DNA-dependent ATPase, and DNA helicase activities. The mutant DnaBbetagamma protein, in which domain alpha was deleted, formed a hexamer; whereas the mutant DnaBalphabeta, in which domain gamma was deleted, could form only dimers. The dimerization of DnaBalphabeta was Mg(2+) dependent. These data suggest that the oligomerization of DnaB helicase involves at least two distinct protein-protein interaction sites; one of these sites is located primarily within domain beta (site 1), while the other interaction site is located within domain gamma (site 2). The mutant DnaBbeta, a polypeptide of 147 aa, where both domains alpha and gamma were deleted, displayed a completely functional ATPase activity. This domain, thus, constitutes the "central catalytic domain" for ATPase activity. The ATPase activity of DnaBalphabeta was kinetically comparable to that of DnaBbeta, indicating that domain alpha had little or no influence on the ATPase activity. In both cases, the ATPase activities were DNA independent. DnaBbetagamma had a DNA-dependent ATPase activity that was kinetically comparable to the ATPase activity of wild-type DnaB protein (wtDnaB), indicating a specific role for C-terminal domain gamma in enhancement of the ATPase activity of domain beta as well as in DNA binding. Mutant DnaBbetagamma, which lacked domain alpha, was devoid of any helicase activity pointing to a significant role for domain alpha. The major findings of this study are (i) domain beta contained a functional ATPase active site; (ii) domain gamma appeared to be the DNA binding domain and a positive regulator of the ATPase activity of domain beta; (iii) although domain alpha did not have any significant effect on the ATPase, DNA binding activities, or hexamer formation, it definitely plays a pivotal role in transducing the energy of ATP hydrolysis to DNA unwinding by the hexamer; and (iv) all three domains are required for helicase activity.     

Nicked-site substrates for a serine recombinase reveal enzyme–DNA communications and an essential tethering role of covalent enzyme–DNA linkages

To analyse the mechanism and kinetics of DNA strand cleavages catalysed by the serine recombinase Tn3 resolvase, we made modified recombination sites with a single-strand nick in one of the two DNA strands. Resolvase acting on these sites cleaves the intact strand very rapidly, giving an abnormal half-site product which accumulates. We propose that these reactions mimic second-strand cleavage of an unmodified site. Cleavage occurs in a synapse of two sites, held together by a resolvase tetramer; cleavage at one site stimulates cleavage at the partner site. After cleavage of a nicked-site substrate, the half-site that is not covalently linked to a resolvase subunit dissociates rapidly from the synapse, destabilizing the entire complex. The covalent resolvase–DNA linkages in the natural reaction intermediate thus perform an essential DNA-tethering function. Chemical modifications of a nicked-site substrate at the positions of the scissile phosphodiesters result in abolition or inhibition of resolvase-mediated cleavage and effects on resolvase binding and synapsis, providing insight into the serine recombinase catalytic mechanism and how resolvase interacts with the substrate DNA.     

Telomere resolution by Borrelia burgdorferi ResT through the collaborative efforts of tethered DNA binding domains

Borrelia burgdorferi, a causative agent of Lyme disease, has a highly unusual segmented genome composed of both circular molecules and linear DNA replicons terminated by covalently closed hairpin ends or telomeres. Replication intermediates of the linear molecules are processed into hairpin telomeres via the activity of ResT, a telomere resolvase. We report here the results of limited proteolysis and mass spectroscopy to identify two main structural domains in ResT, separated by a chymotrypsin cleavage site between residues 163 and 164 of the 449 amino acid protein. The two domains have been overexpressed and purified. DNA electrophoretic mobility shift assays revealed that the C-terminal domain (ResT(164-449)) displays sequence-specific DNA binding to the box 3,4,5 region of the telomere, while the N-terminal domain (ResT(1-163)) exhibits sequence-independent DNA binding activity. Further analysis by DNase I footprinting supports a model for telomere resolution in which the hairpin binding module of the N-terminal domain is delivered to the box 1,2 region of the telomere through its tethering to ResT(164-449). Conversely, ResT(1-164) may play an important regulatory role by modulating both sequence-specific DNA binding activity and catalysis by the C-terminal domain.     

Separation of the complex DNA binding domain of EBNA-1 into DNA recognition and dimerization subdomains of novel structure.

EBNA-1 is essential for replication of the latent episomal form of the Epstein-Barr virus genome and is involved in regulation of viral latency promoters. EBNA-1 activity is mediated through direct DNA binding. The DNA binding and dimerization functions of EBNA-1 have previously been located to a carboxy-terminal domain, amino acids (aa) 459 to 607. To identify and define the subdomains for these two functions, we created an extensive series of deletions and point mutations in an EBNA-1 (aa 408 to 641) background. The ability of the EBNA-1 mutants to heterodimerize with a wild-type EBNA-1 (aa 459 to 641) Immunoprecipitation assays with a monoclonal antibody, EBNA.OT1x, that recognizes EBNA-1 (aa 408 to 641) but not EBNA-1 (aa 459 to 641). These experiments revealed that mutations affecting dimerization occurred over two separate regions, aa 501 to 532 and aa 554 to 598. DNA binding was tested in mobility shift assays against a panel of oligonucleotide-binding sites. Dimerization was a prerequisite for DNA binding. The DNA recognition domain was localized to a separate region, aa 459 to 487, upstream of the dimerization domain. EBNA-1 variants carrying substitutions at aa 467 and 468 and at aa 477 gave a pattern of binding to mutant oligonucleotide probes that implicates these particular amino acids in DNA recognition. EBNA-1 appears to utilize novel mechanisms for both DNA recognition and dimerization since neither domain conforms to previously described structural motifs.