Beta Recombinase Research Articles

Functionality of the β/six Site-Specific Recombination System in Tobacco and Arabidopsis: A Novel Tool for Genetic Engineering of Plant Genomes

The beta recombinase is a member of the prokaryotic site-specific serine recombinases (invertase/resolvase family), which in the presence of a DNA bending cofactor can catalyse DNA deletions between two directly oriented 90-bp six recombination sites. We have examined here whether the beta recombinase can be expressed in plants and whether it displays in planta its specific catalytic activity excising DNA sequences that are flanked by six sites. In plant protoplasts, the enzyme could be expressed as a GFP-beta recombinase fusion which can localise to the cell nucleus. Beta recombinase stably expressed in tobacco plants can catalyse deletion of a spacer region that is flanked by directly oriented six sites and has been placed between promoter and a GUS reporter gene (preventing GUS expression). In transient transformation experiments, beta recombinase-mediated elimination of the spacer results in transcriptional induction of the GUS gene. Similarly, beta recombinase in stably double-transformed Arabidopsis plants deletes specifically the spacer region of a reporter construct that has been incorporated into the genome. In the segregating T1 generation, plants were identified that contain exclusively the recombined reporter construct. In summary, our results demonstrate that functional / recombinase can be expressed in plants and that the enzyme is suitable to precisely eliminate undesired sequences from plant genomes. Therefore, the beta/six recombination system (and presumably related recombinases) may become an attractive tool for plant genetic engineering.

Conformation and stability of the Streptococcus pyogenes pSM19035-encoded site-specific β recombinase, and identification of a folding intermediate

Solution properties of beta recombinase were studied by circular dichroism and fluorescence spectroscopy, size exclusion chromatography, analytical ultracentrifugation, denaturant-induced unfolding and thermal unfolding experiments. In high ionic strength buffer (1 M NaCl) beta recombinase forms mainly dimers, and strongly tends to aggregate at ionic strength lower than 0.3 M NaCl. Urea and guanidinium chloride denaturants unfold beta recombinase in a two-step process. The unfolding curves have bends at approximately 5 M and 2.2 M in urea and guanidinium chloride-containing buffers. Assuming a three-state unfolding model (N2-->2I-->2U), the total free energy change from 1 mol of native dimers to 2 mol of unfolded monomers amounts to deltaG(tot) = 17.9 kcal/mol, with deltaG(N2-->2I) = 4.2 kcal/mol for the first transition and deltaG(I-->U) = 6.9 kcal/mol for the second transition. Using sedimentation-equilibrium analytical ultracentrifugation, the presence of beta recombinase monomers was indicated at 5 M urea, and the urea dependence of the circular dichroism at 222 nm strongly suggests that folded monomers represent the unfolding intermediate.

Synapsis and strand exchange in the resolution and DNA inversion reactions catalysed by the beta recombinase.

In the presence of a sequence-independent chromatin-associated protein, such as Hbsu or HMGB, the beta recombinase catalyses resolution between two directly oriented recombination sites (six sites) and both resolution and DNA inversion between two inversely oriented six sites. Assembly of the synaptic complex requires binding of the beta recombinase to the six sites and the presence of Hbsu. Whether resolution or inversion will take place depends on the relative orientation of the two six sites, the level of DNA supercoiling and the amounts of Hbsu. In this work, the topologies of the products of the resolution and inversion reactions were analysed. The resolution reaction generated mainly singly catenated DNA circles, while DNA inversion gave rise to unknotted circles and small amounts of DNA molecules containing 3- or 5-noded knots. In spite of the distinctive features of the beta system, the topology of synapsis and strand exchange during the resolution reaction is similar to that of Tn3 and gammadelta resolvases. The ability of the beta recombinase to catalyse both inversion and resolution reactions probably reflects different possible architectures of the synaptic complex, which to a large extent depends on Hbsu.

Plant chromosomal HMGB proteins efficiently promote the bacterial site-specific beta-mediated recombination in vitro and in vivo.

In the presence of an accessory DNA bending protein, the bacterial site-specific beta recombinase catalyzes resolution and DNA inversion. Five different maize high mobility group B (HMGB) proteins were examined for their potential to facilitate beta recombination in vitro using DNA substrates with different intervening distances (73-913 bp) between two directly oriented recombination (six) sites. All analyzed HMGB proteins (HMGB1 to HMGB5) could promote beta recombination, but depending on the DNA substrate with different efficiencies. The HMGB1 protein displayed an activity comparable to that of the natural promoting protein Hbsu, whereas the other HMGB proteins were less effective. Phosphorylation of the HMGB1 protein resulted in an increased efficiency of HMGB1 to promote beta recombination. Analyses of DNA substrates with closely spaced six sites demonstrated that in the presence of HMGB1 the recombination rate was correlated to the distance between the six sites, but independent of the helical orientation of the six sites. Using a Bacillus subtilis strain defective in Hbsu, the coexpression of beta recombinase and HMGB1 (or a truncated HMGB1 derivative) revealed that a plant HMG-box domain protein is sufficient for assisting beta to catalyze recombination in vivo. Our results using beta recombination as a model system suggest that the various plant HMGB proteins (and their posttranslationally modified versions) have the potential of forming a repertoire of different DNA structures, which is compatible with the idea that the HMGB proteins can act as architectural factors in a variety of nucleoprotein structures.

Proteolytic cleavage of gram-positive beta recombinase is required for crystallization.

Beta recombinase, a DNA resolvase-invertase, catalyzes in the presence of a chromatin-associated protein such as Hbsu, DNA resolution or DNA inversion on supercoiled substrates containing two directly or inversely oriented target (six) sites. Single crystals of the beta recombinase from plasmid pSM19035 were obtained using the vapor diffusion technique with ammonium phosphate as the precipitating agent. The crystals diffracted X-rays to a maximum resolution of 2.5A. Due to proteolytic degradation during the crystallization experiment, the crystals contain only the N-terminal catalytic domain of beta recombinase corresponding to about 60% of the molecular mass of the initially assayed native protein. The proteolytic removal of the C-terminal DNA-binding domain demonstrated that protein modification can be essential to provide material suitable for X-ray analysis.

β Recombinase Catalyzes Inversion and Resolution between Two Inversely Oriented six Sites on a Supercoiled DNA Substrate and Only Inversion on Relaxed or Linear Substrates

The beta recombinase, in the presence of a chromatin-associated protein such as Hbsu, catalyzes DNA resolution or DNA inversion on supercoiled substrates containing two directly or inversely oriented six sites. Hbsu stabilizes the formation of the recombination complex (Alonso, J. C., Weise, F., and Rojo, F. (1995) J. Biol. Chem. 270, 2938-2945). In this study we show that resolution by beta recombinase strictly requires supercoiled DNA, but inversion does not. On a substrate with two inversely oriented six sites, beta recombinase catalyzed both resolution and inversion if the DNA was supercoiled but only inversion if the substrate was relaxed or linear. Hbsu was critical for the formation of synaptic complexes; its concentration relative to that of the supercoiled DNA substrate determined whether resolution or inversion products were preferentially formed. The results suggest that the beta recombinase forms unproductive short-lived synaptic complexes between two juxtaposed inversely oriented six sites; the presence of 3 to 13 Hbsu dimers per supercoiled DNA molecule would stabilize a synaptic complex with a relative geometry of the six sites allowing beta recombinase preferentially to achieve resolution. Supercoiling probably helps to overcome an energetic barrier, since resolution does not occur in relaxed DNA. The presence of >30 Hbsu dimers per DNA molecule probably favors the formation of a recombination complex with a different geometry since the reaction is directed preferentially toward DNA inversion.

Mutational analysis of a site-specific recombinase: characterization of the catalytic and dimerization domains of the beta recombinase of pSM19035.

The beta recombinase encoded by the streptococcal plasmid pSM19035, which shows 28 to 34% identity with DNA resolvases and DNA invertases, can catalyze formation of deletions or inversions between properly oriented target sites. We have constructed a number of site-directed mutations at residues that are conserved between the beta protein and other DNA recombinases of the resolvase/invertase family. The analysis of the recombination and DNA-binding ability of each mutant protein shows that the mutations affect the catalytic activity and, in two cases, the dimerization of the protein. The results suggest that the beta protein probably mediates recombination by a catalytic mechanism similar to that proposed for the resolvase/invertase family. Since the beta recombinase differs from DNA resolvases and DNA invertases in its lack of bias towards either of these reactions, the results presented support the hypothesis that its unique properties might depend on details of the architecture or assembly of the recombination complex. In addition, two beta protein mutants that can no longer form dimers in solution have provided new insights into the way the protein binds to DNA.

The role of chromatin-associated protein Hbsu in beta-mediated DNA recombination is to facilitate the joining of distant recombination sites.

The beta recombinase is unable to mediate in vitro DNA recombination between two directly oriented recombination sites unless a bacterial chromatin-associated protein (Bacillus subtilis Hbsu or Escherichia [correction of Eschrichia] coli HU] is provided. By electron microscopy, we show that the role of Hbsu is to help in joining the recombination sites to form a stable synaptic complex. Some evidence supports the fact that Hbsu works by recognizing and stabilizing a DNA structure at the recombination site, rather than by serving as a bridge between beta recombinase dimers through a protein-protein interaction. We show that the mammalian HMG1 protein, which shares neither sequence nor structural homology with Hbsu, can also stimulate beta-mediated recombination. These chromatin-associated proteins share the property of binding to DNA in a relatively non-specific fashion, bending it, and having a marked preference for altered DNA structures. Hbsu, HU or HMG1 proteins probably bind specifically at the crossing-over region, since at limiting protein-DNA molar ratios they could not be outcompeted by an excess of a DNA lacking the crossing over site. Distamycin, a minor groove binder that induces local distortions in DNA, did not affect the binding of beta protein to DNA, but inhibited the formation of the synaptic complex.

The Bacillussubtilis Histone-like Protein Hbsu Is Required for DNA Resolution and DNA Inversion Mediated by the β Recombinase of Plasmid pSM19035

The beta recombinase, encoded by the Gram-positive bacterial plasmid pSM19035, is unable to mediate DNA recombination in vitro unless a host factor is provided. The factor has now been identified as the Bacillus subtilis Hbsu protein. Hbsu is a nonspecific DNA-binding and DNA-bending protein. The beta recombinase, in the presence of highly purified Hbsu protein, is able to catalyze in vitro intramolecular recombination between two specific recombination sites on a supercoiled DNA molecule. DNA resolution was obtained when the two crossing over sites (six sites) were directly oriented, whereas DNA inversion was the product when the six sites were in inverse orientation. The ability of the Escherichia coli chromatin-associated proteins HU, IHF, Fis, and H-NS to substitute for Hbsu was investigated. HU efficiently stimulated beta-mediated recombination, while the effect of IHF was partial and that of Fis and H-NS was undetectable. In addition, the beta protein was able to mediate DNA recombination in both wild-type and IHF-deficient E. coli cells, but failed to do so in an HU-deficient strain. The data presented provide direct evidence that a chromatin-associated protein is strictly required for beta-mediated recombination.

The beta recombinase of plasmid pSM19035 binds to two adjacent sites, making different contacts at each of them.

The beta recombinase from plasmid pSM19035 catalyzes intramolecular site-specific recombination between two directly or inversely oriented six sites in the presence of a chromatin-associated protein (Hbsu, HU or HMG-1). The six site is a DNA segment containing two binding sites (I and II) for beta protein dimers. We show that beta recombinase binds sequentially to both sites, having a different affinity for each one. Hydroxyl radical footprints show a different protection pattern at each site. Positions critical for beta protein binding have been identified by methylation interference and missing nucleoside assays. The results indicate that the protein recognizes each site in a different way. Comparison of the beta protein recombination site with that of DNA resolvases and DNA invertases of the Tn3 family, to which it belongs, shows that these sequences can be divided into two regions. One corresponds to the crossover point and is similar for all recombinases of the family. The other region differs in the different subfamilies and seems to have an architectural role in aligning the crossover sites at the synaptic complex. The different ways to assemble this complex could explain why each system leads to a particular recombination event: DNA resolution (resolvases), inversion (invertases) or both (beta recombinase).

The beta recombinase from the Streptococcal plasmid pSM 19035 represses its own transcription by holding the RNA polymerase at the promoter region.

The beta protein encoded by the Streptococcus pyogenes plasmid pSM19035 is a site-specific recombinase involved in both resolution of plasmid multimers into monomers and DNA inversion. It has been proposed that the DNA region to which the beta recombinase binds to mediate recombination includes a promoter from which orf alpha and the beta gene are transcribed. We have determined the sites at which transcription of the orf alpha and the beta gene initiates in vitro and we have demonstrated that highly purified beta recombinase acts as a repressor of its own synthesis. The promoters are located within the beta recombinase binding site, which we have defined previously. The binding of the beta recombinase to its target site does not seem to exclude RNA polymerase from the promoter, despite the overlapping of their binding sites. Therefore, it is likely that the beta recombinase does not repress transcription by a mere steric hindrance on RNA polymerase binding.