Stepwise dissection of the Hin-catalyzed recombination reaction from synapsis to resolution

The Hin DNA invertase promotes a site-specific DNA recombination reaction in the Salmonella chromosome. The native Hin reaction exhibits overwhelming selectivity for promoting inversions between appropriately oriented recombination sites and requires the Fis regulatory protein, a recombinational enhancer, and a supercoiled DNA substrate. Here, we report a robust recombination reaction employing oligonucleotide substrates and a hyperactive mutant form of Hin. Synaptic complex intermediates purified by gel electrophoresis were found to contain four Hin protomers bound to two recombination sites. Each Hin protomer is associated covalently with a cleaved DNA end. The cleaved complexes can be ligated into both parental and recombinant orientations at equivalent frequencies, provided the core residues can base-pair, and are readily disassembled into separated DNA fragments bound by Hin dimers. Kinetic analyses reveal that synapsis occurs rapidly, followed by comparatively slow Hin-catalyzed DNA cleavage. Subsequent steps of the reaction, including DNA exchange and ligation, are fast. Thus, post-synaptic step(s) required for DNA cleavage limit the overall rate of the recombination reaction.     

Intrasubunit and intersubunit interactions controlling assembly of active synaptic complexes during Hin-catalyzed DNA recombination

Serine recombinases, which generate double-strand breaks in DNA, must be carefully regulated to ensure that chemically active DNA complexes are assembled correctly. In the Hin-catalyzed site-specific DNA inversion reaction, two inversely oriented recombination sites on the same DNA molecule assemble into a synaptic complex that uniquely generates inversion products. The Fis-bound recombinational enhancer, together with topological constraints directed by DNA supercoiling, functions to regulate Hin synaptic complex formation and activity. We have isolated a collection of gain-of-function mutants in 22 positions within the catalytic and oligomerization domains of Hin using two genetic screens and by site-directed mutagenesis. One genetic screen measured recombination in the absence of Fis and the other assessed SOS induction as a readout of increased DNA cleavage. These mutations, together with molecular modeling, identify important sites of dynamic intrasubunit and intersubunit interactions that regulate assembly of the active tetrameric recombination complex. Of particular interest are interactions between the oligomerization helix (helix E) and the catalytic domain of the same subunit that function to hold the dimer in an inactive state in the absence of the Fis/enhancer system. Among these is a relay involving a triad of phenylalanines that are proposed to switch positions during the transition from dimers to the catalytically active tetramer. Novel Hin mutants that generate synaptic complexes that are blocked at steps prior to DNA cleavage are also described.     

Topological analysis of Hin-catalysed DNA recombination in vivo and in vitro

In vitro studies have demonstrated that Hin-catalysed site-specific DNA inversion occurs within a tripartite invertasome complex assembled at a branch on a supercoiled DNA molecule. Multiple DNA exchanges within a recombination complex (processive recombination) have been found to occur with particular substrates or reaction conditions. To investigate the mechanistic properties of the Hin recombination reaction in vivo, we have analysed the topology of recombination products generated by Hin catalysis in growing cells. Recombination between wild-type recombination sites in vivo is primarily limited to one exchange. However, processive recombination leading to knotted DNA products is efficient on substrates containing recombination sites with non-identical core nucleotides. Multiple exchanges are limited by a short DNA segment between the Fis-bound enhancer and closest recombination site and by the strength of Fis-Hin interactions, implying that the enhancer normally remains associated with the recombining complex throughout a single exchange reaction, but that release of the enhancer leads to multiple exchanges. This work confirms salient mechanistic aspects of the reaction in vivo and provides strong evidence for the propensity of plectonemically branched DNA in prokaryotic cells. We also demonstrated that a single DNA exchange resulting in inversion in vitro is accompanied by a loss of four negative supercoils.     

Hin-mediated site-specific recombination requires two 26 bp recombination sites and a 60 bp recombinational enhancer

The alternate expression of flagellin genes in Salmonella is the result of an inversion of a 996 bp segment of chromosomal DNA. We have analyzed the components of this site-specific recombination reaction in an in vitro system derived from E. coli. Efficient Hin-mediated inversion requires the 20,000 MW Hin protein and a proteinase K-sensitive host component. The supercoiled DNA substrate must contain two 26 bp recombination sites in inverted configuration and a 60 bp sequence that increases the rate of recombination over 20-fold. This recombinational enhancer can function at many different locations and consists of at least two noncontiguous sequence domains whose relative orientation, but not precise spacing, with respect to each other is important. Synthetically derived wild-type and mutant recombination sites were constructed to analyze the sequence and structural features that are important within the recombination site.     

Host protein requirements for in vitro site-specific DNA inversion

Flagellar phase variation is mediated by a recombination event that occurs at specific sites leading to inversion of a chromosomal segment of DNA. The presence of a 60 bp recombinational enhancer sequence on the DNA substrate molecule results in a 150-fold stimulation in the initial rate of inversion. The protein components required for inversion have been purified. They include the 21,000 dalton recombinase (Hin), a 12,000 dalton host protein (Factor II), and one of the major histone-like proteins of E. coli HU. The dependence of the initial rate of recombination on HU varies with respect to the location of the recombinational enhancer. The role of HU, Factor II, and the enhancer in facilitating site-specific recombination is discussed.     

Role of arginine-43 and arginine-69 of the Hin recombinase catalytic domain in the binding of Hin to the hix DNA recombination sites

The Hin recombinase mediates the site-specific inversion of a segment of the Salmonella chromosome between two flanking 26 bp hix DNA recombination sites. Mutations in two amino acid residues, R43 and R69 of the catalytic domain of the Hin recombinase, were identified that can compensate for loss of binding resulting from elimination of certain major and minor groove contacts within the hix recombination sites. With one exception, the R43 and R69 mutants were also able to bind a hix sequence with an additional 4 bp added to the centre of the site, unlike wild-type Hin. Purified Hin mutants R43H and R69C had both partial cleavage and inversion activities in vitro while mutants R43L, R43C, R69S, and R69P had no detectable cleavage and inversion activities. These data support a model in which the catalytic domain plays a role in DNA-binding specificity, and suggest that the arginine residues at positions 43 and 69 function to position the Hin recombinase on the DNA for a step in the recombination reaction which occurs either at and/or prior to DNA cleavage.     

Multiple DNA binding activities of the novel site-specific recombinase, Piv, from Moraxella lacunata

The recombinase, Piv, is essential for site-specific DNA inversion of the type IV pilin DNA segment in Moraxella lacunata and Moraxella bovis. Piv shows significant homology with the transposases of the IS110/IS492 family of insertion elements, but, surprisingly, Piv contains none of the conserved amino acid motifs of the lambda Int or Hin/Res families of site-specific recombinases. Therefore, Piv may mediate site-specific recombination by a novel mechanism. To begin to determine how Piv may assemble a synaptic nucleoprotein structure for DNA cleavage and strand exchange, we have characterized the interaction of Piv with the DNA inversion region of M. lacunata. Gel shift and nuclease/chemical protection assays, competition and dissociation rate analyses, and cooperativity studies indicate that Piv binds two distinct recognition sequences. One recognition sequence, found at multiple sites within and outside of the invertible segment, is bound by Piv protomers with high affinity. The second recognition sequence is located at the recombination cross-over sites at the ends of the invertible element; Piv interacts with this sequence as an oligomer with apparent low affinity. A model is proposed for the role of the different Piv binding sites of the M. lacunata inversion region in the formation of an active synaptosome.     

Effect of DNA superhelicity and bound proteins on mechanistic aspects of the Hin-mediated and Fis-enhanced inversion

Using a recently developed inhomogeneous, macroscopic model for long DNA bound to proteins, we examine topological and geometric aspects of DNA/protein structures and dynamics on various stages of the Hin inversion pathway. This biological reaction involves exchange of DNA in a synaptic complex that brings together several DNA sites bound to Hin dimers as well as Fis enhancers. Brownian dynamics simulations in the millisecond timescale allow us to follow and analyze the DNA/protein dynamics trajectories and to examine the effects of DNA superhelicity and protein binding on various reaction steps. Analysis of the generated kinetic pathways helps explain mechanistic aspects regarding the process by which two or three protein-bound DNA sites come to close spatial proximity and show that how topological selectivity (two trapped supercoils), enhancer binding, and properties of supercoiled DNA play critical roles in regulating the inversion reaction. Specifically, a critical amount of DNA superhelicity (e.g., |sigma| > 0.02) leads to an optimal interplay for the first reaction step-two-site juxtaposition-between large-scale random rearrangements of Hin-bound DNA and local slithering within branches of plectonemes. The three-site juxtaposition, the second step, is significantly accelerated by the presence of an enhancer protein that, due to severe local bending, also alters juxtaposition mechanisms, especially for superhelical density magnitude greater than around 0.04.     

DNA looping and the helical repeat in vitro and in vivo: effect of HU protein and enhancer location on Hin invertasome assembly.

Site-specific DNA inversion by the Hin recombinase requires the formation of a multicomponent nucleo-protein structure called an invertasome. In this structure, the two recombination sites bound by Hin are assembled together at the Fis-bound recombinational enhancer with the requisite looping of the intervening DNA segments. We have analyzed the role of the HU protein in invertasome assembly when the enhancer is located at variable positions close to one of the recombination sites. In the absence of HU in vitro and in hupA hupB mutant cells in vivo, invertasome assembly is very inefficient when there is < 104 bp of DNA between the enhancer and recombination site. Invertasome assembly in the presence of HU in vitro or in vivo displayed a periodicity beginning with 60 bp of intervening DNA that reflected its helical repeat. The average helical repeat for this DNA region was calculated by autocorrelation and Fourier transformation to be 11.2 bp per turn for supercoiled DNA both in the presence of HU in vitro and in hup+ cells in vivo. HU is the only protein in Escherichia coli that can promote invertasome formation with short DNA lengths between the enhancer and recombination sites. However, the presence of certain polyamines and a protein activity present in HeLa nuclear extracts can efficiently substitute for HU in invertasome assembly. These data support a model in which HU binds non-specifically to the DNA between the enhancer and recombination site to facilitate DNA looping.     

DNA inversions in phages and bacteria

In certain phages and bacteria, there is a recombination system that specifically promotes the inversion of a DNA fragment. These inversion events appear to act as genetic switches allowing the alternate expression of different sets of genes which in general code for surface proteins. The mechanism of inversion in one class of inversion systems (Gin/Hin) has been studied in detail. It involves the formation of a highly specific nucleoprotein complex in which not only the two recombination sites and the DNA invertase participate but also a recombinational enhancer to which the DNA-bending protein Fis is bound.     

The recombinational enhancer for DNA inversion functions independent of its orientation as a consequence of dyad symmetry in the Fis-DNA complex.

The Escherichia coli Fis protein binds to specific DNA sequences whose base composition varies enormously. One known function of Fis is to stimulate site-specific DNA recombination. We used the Gin-mediated DNA inversion system of bacteriophage Mu to analyze Fis-DNA interaction. Efficient inversion requires an enhancer which consists of two Fis binding sites at a fixed distance from each other. Using mutant enhancers in which one of the Fis binding sites is replaced we show that Fis binds symmetrically to the DNA and we locate the center of symmetry. Furthermore, we show that one of the Fis binding sites can be replaced by a Fis binding site that normally functions in a process other than site-specific recombination.     

Purification and DNA-binding properties of FIS and Cin, two proteins required for the bacteriophage P1 site-specific recombination system, cin

An Escherichia coli chromosomally coded factor termed FIS (Factor for Inversion Stimulation) stimulates the Cin protein-mediated, site-specific DNA inversion system of bacteriophage P1 more than 500-fold. We have purified FIS and the recombinase Cin, and studied the inversion reaction in vitro. DNA footprinting studies with DNase I showed that Cin specifically binds to the recombination site, called cix. FIS does not bind to cix sites but does bind to a recombinational enhancer sequence that is required in cis for efficient recombination. FIS also binds specifically to sequences outside the enhancer, as well as to sequences unrelated to Cin inversion. On the basis of these data, we discuss the possibility of additional functions for FIS in E. coli.     

Real-time single-molecule tethered particle motion experiments reveal the kinetics and mechanisms of Cre-mediated site-specific recombination

Tyrosine family recombinases (YRs) are widely utilized in genome engineering systems because they can easily direct DNA rearrangement. Cre recombinases, one of the most commonly used types of YRs, catalyze site-specific recombination between two loxP sites without the need for high-energy cofactors, other accessory proteins or a specific DNA target sequence between the loxP sites. Previous structural, analytical ultracentrifuge and electrophoretic analyses have provided details of the reaction kinetics and mechanisms of Cre recombinase activity; whether there are reaction intermediates or side pathways involved has been left unaddressed. Using tethered particle motion (TPM), the Cre-mediated site-specific recombination process has been delineated, from beginning to end, at the single-molecule level, including the formation of abortive complexes and wayward complexes blocking inactive nucleoprotein complexes from entering the recombination process. Reversibility in the strand-cleavage/-ligation process and the formation of a thermally stable Holliday junction intermediate were observed within the Cre-mediated site-specific recombination process. Rate constants for each elementary step, which explain the overall reaction outcomes under various conditions, were determined. Taking the findings of this study together, they demonstrate the potential of single-molecule methodology as an alternative approach for exploring reaction mechanisms in detail.     

Sequence-specific interaction of the Salmonella Hin recombinase in both major and minor grooves of DNA.

The Hin recombinase of Salmonella catalyzes a site-specific recombination event which leads to flagellar phase variation. Starting with a fully symmetrical recombination site, hixC, a set of 40 recombination sites which vary by pairs of single base substitutions was constructed. This set was incorporated into the Salmonella-specific bacteriophage P22 based challenge phage selection and used to define the DNA sequence determinants for the binding of Hin to DNA in vivo. The critical sequence-specific contacts between a Hin monomer and a 13 bp hix half-site are at two T:A base pairs in the major groove of the DNA which are separated by one base pair, and two consecutive A:T contacts in the minor groove. The base substitutions in the major groove recognition portion which were defective in binding Hin still retained residual binding capability in vivo, while the base pair substitutions affecting the minor groove recognition region lost all in vivo binding. Using in vitro binding assays, Hin was found to bind to hix symmetrical sites with A:T base pairs or I:C base pairs in the minor groove recognition sequences, but not to G:C base pairs. In separate in vitro binding assays, Hin was equally defective in binding to either a G:C or a I:C contact in a major groove recognition sequence. Results from in vitro binding assays to hix sites in which 3-deazaadenine was substituted for adenine are consistent with Hin making a specific contact to either the N3 of adenine or O2 of thymine in the minor groove within the hix recombination site on each symmetric half-site. These results taken with the results of previous studies on the DNA binding domain of Hin suggest a sequence-specific minor groove DNA binding motif.     

The DNA invertase Gin of phage Mu: formation of a covalent complex with DNA via a phosphoserine at amino acid position 9.

The DNA invertase Gin encoded by bacteriophage Mu catalyses efficient site-specific recombination between inverted repeat sequences (IR) in vivo and in vitro in the presence of the host factor FIS and the recombinational enhancer. We demonstrate that Gin alone is able to introduce single strand breaks into duplex DNA fragments which contain the IR sequence. Strand cleavage is site-specific and can occur on either strand within the IR. Cleaved molecules contain Gin covalently attached to DNA. The covalent complex is formed through linkage of Gin to the 5' DNA phosphate at the site of the break via a phosphoserine. Extensive site-directed mutational analysis showed that all mutants altered at serine position 9 were completely recombination deficient in vivo and in vitro. The mutant proteins bind to DNA but lack topoisomerase activity and are unable to introduce nicks. This holds true even for a conservative amino acid substitution at position 9. We conclude that serine at position 9 is part of the catalytic domain of Gin. The intriguing finding that the DNA invertase Gin has the same catalytic center as the DNA resolvases that promote deletions without recombinational enhancer and host factor FIS is discussed.     

The mu transpososome through a topological lens

Phage Mu is the most efficient transposable element known, its high efficiency being conferred by an enhancer DNA element. Transposition is the end result of a series of well choreographed steps that juxtapose the enhancer and the two Mu ends within a nucleoprotein complex called the 'transpososome.' The particular arrangement of DNA and protein components lends extraordinary stability to the transpososome and regulates the frequency, precision, directionality, and mechanism of transposition. The structure of the transpososome, therefore, holds the key to understanding all of these attributes, and ultimately to explaining the runaway genetic success of transposable elements throughout the biological world. This review focuses on the path of the DNA within the Mu transpososome, as uncovered by recent topological analyses. It discusses why Mu topology cannot be analyzed by standard methods, and how knowledge of the geometry of site alignment during Flp and Cre site-specific recombination was harnessed to design a new methodology called 'difference topology.' This methodology has also revealed the order and dynamics of association of the three interacting DNA sites, as well as the role of the enhancer in assembly of the Mu transpososome.     

Two DNA invertases contribute to flagellar phase variation in Salmonella enterica serovar Typhimurium strain LT2

Salmonella enterica serovar Typhimurium strain LT2 possesses two nonallelic structural genes, fliC and fljB, for flagellin, the component protein of flagellar filaments. Flagellar phase variation occurs by alternative expression of these two genes. This is controlled by the inversion of a DNA segment, called the H segment, containing the fljB promoter. H inversion occurs by site-specific recombination between inverted repetitious sequences flanking the H segment. This recombination has been shown in vivo and in vitro to be mediated by a DNA invertase, Hin, whose gene is located within the H segment. However, a search of the complete genomic sequence revealed that LT2 possesses another DNA invertase gene that is located adjacent to another invertible DNA segment within a resident prophage, Fels-2. Here, we named this gene fin. We constructed hin and fin disruption mutants from LT2 and examined their phase variation abilities. The hin disruption mutant could still undergo flagellar phase variation, indicating that Hin is not the sole DNA invertase responsible for phase variation. Although the fin disruption mutant could undergo phase variation, fin hin double mutants could not. These results clearly indicate that both Hin and Fin contribute to flagellar phase variation in LT2. We further showed that a phase-stable serovar, serovar Abortusequi, which is known to possess a naturally occurring hin mutation, lacks Fels-2, which ensures the phase stability in this serovar.     

Enhancer-independent mutants of the Cin recombinase have a relaxed topological specificity.

Cin is a member of the hin family of complementing site-specific recombinases which regulate the alternate expression of genes by inverting DNA segments. Common characteristics of this family of recombination systems are the requirement for an enhancer-like element in cis and the specificity for inversely oriented recombination sites on the same DNA molecule. We have isolated two mutants of the Cin recombinase which will efficiently recombine a substrate lacking the enhancer. In addition, these mutant proteins also catalyse efficient recombination between sites in direct orientation or on different DNA molecules. Both mutations are due to single amino acid substitutions at different positions in the protein and the two mutants have slightly different phenotypes. The finding that the loss of enhancer dependence is coupled to a change in topological specificity leads us to conclude that the enhancer determines the specificity of the system for DNA inversion.     

Multi-site-specific endonucleases and the initiation of homologous genetic recombination in yeast

The notion that homologous recombination is a regulated biological process is not a familiar one. In yeasts, homologous recombination and most site-specific ones are initiated by site-specific double-stranded breaks that are introduced within cis-acting elements for the recombination. On the other hand, yeasts have a group of site-specific endonucleases (multi-site-specific endonucleases) that have a number of cleavage sites on each DNA. One of them, Endo.SceI of S. cerevisiae, was shown to introduce double-stranded breaks at a number of welldefined sites on the mitochondrial DNA in vivo. An Endo.SceI-induced double-stranded break was demonstrated to induce homologous recombination in mitochondria. Like the case of homologous recombination of nuclear chromosomes, the double-stranded break induces gene conversion of both genetic markers flanking and in the proximity of the cleavage site, and the cleaved DNA acts as a recipient of genetic information from the uncleaved partner DNA. The 70 kDa-heat-shock protein (HSP70)-subunit of Endo.SceI and a general role of the HSP70 in the regulation of protein-folding suggest the regulation of nucleolytic activity of Endo.SceI.     

Expression of the bacteriophage P1 cin recombinase gene from its own and heterologous promoters

The cin recombinase of bacteriophage P1, a protein that catalyses site-specific DNA inversions, has been identified and its structural gene has been cloned under the control of different promoters. One of the DNA sequences used for the site-specific recombination, cixL, overlaps with the 3' end of the gene, but we show that the presence of this site does not affect cin gene expression from strong promoters. To assay cin activity we have constructed plasmids that carry antibiotic resistance genes within the invertible segment that are transcribed from promoters outside the segment. DNA inversion switches on or off genes for chloramphenicol or kanamycin resistance. These tester plasmids are used to study cin-mediated DNA inversion both in vivo and in vitro.