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1.
The site-specific inversion reaction controlling flagellin synthesis in Salmonella involves the function of three proteins: Hin, Fis and HU. The DNA substrate must be supercoiled and contain a recombinational enhancer sequence in addition to the two recombination sites. Using mutant substrates or modified reaction conditions, large amounts of complexes can be generated which are recognized by double-stranded breaks within both recombination sites upon quenching. The cleaved molecules contain 2-bp staggered cuts within the central dinucleotide of the recombination site. Hin is covalently associated with the 5' end while the protruding 3' end contains a free hydoxyl. We demonstrate that complexes generated in the presence of an active enhancer are intermediates that have advanced past the major rate limiting step(s) of the reaction. In the absence of a functional enhancer, Hin is also able to assemble and catalyze site-specific cleavages within the two recombination sites. However, these complexes are kinetically distinct from the complexes assembled with a functional enhancer and cannot generate inversion without an active enhancer. The results suggest that strand exchange leading to inversion is mediated by double-stranded cleavage of DNA at both recombination sites followed by the rotation of strands to position the DNA into the recombinant configuration. The role of the enhancer and DNA supercoiling in these reactions is discussed.  相似文献   

2.
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.  相似文献   

3.
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.  相似文献   

4.
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.  相似文献   

5.
R C Johnson  M I Simon 《Cell》1985,41(3):781-791
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.  相似文献   

6.
The Gin DNA invertase of bacteriophage Mu carries out processive recombination in which multiple rounds of exchange follow synaptic complex formation. The stereostructure of the knotted products determined by electron microscopy establishes critical features of site synapsis and DNA exchange. Surprisingly, the invertase knots substrates with directly repeated sites as well as those with inverted sites. The results suggest that the Gin synaptic complex contains three mutually perpendicular dyads; one is the axis of site rotation during exchange, and they cause inverted and direct site substrates to form a similar synaptic complex. The extensive knotting by Gin has implications for the energetics of recombination and shows that the enhancer for recombination is required only at an early stage, and thus may normally operate in a hit-and-run fashion.  相似文献   

7.
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.  相似文献   

8.
Host protein requirements for in vitro site-specific DNA inversion   总被引:55,自引:0,他引:55  
R C Johnson  M F Bruist  M I Simon 《Cell》1986,46(4):531-539
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.  相似文献   

9.
Phage lambda integrative and excisive recombination normally proceeds by a pair of sequential strand exchanges. During the first exchange reaction, the "top" strand in each recombination site is cleaved, exchanged, and religated generating a Holliday junction intermediate. This intermediate DNA structure is resolved through a pair of reciprocal "bottom" strand exchanges, leading to recombinant products. The strict co-ordination of exchange reactions ensures religation between correct partner strands only. Here we show that the directionality of recombination is altered in vivo by two mutant integrases, Int-h (E174 K) and a double mutant Int-h/218 (E174 K/E218 K). This change in directionality leads to deletion instead of inversion on substrates that carry inverted attachment sites and, depending on the pair of target sites employed, requires the presence or absence of integration host factor. Neither Fis nor Xis is involved in deletion. Sequence analyses of deletion products reveal that the newly generated hybrid attachment site exhibits a reversed genetic polarity. We demonstrate that only one of two possible hybrid site configurations is generated and discuss two pathways leading to deletion. In the first, deletion results from a wrong alignment of the two recombination sites within the synaptic complex. In the second pathway, the unco-ordinated cleavage by the mutant integrases of all four DNA strands present in a conventional Holliday junction intermediate leads to two double-stranded breaks, whereby the subsequent rejoining between "wrong" partner strands appears restricted to only two strands.  相似文献   

10.
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.  相似文献   

11.
12.
An artificial recombination site hixC composed of two identical half-sites that bind the Hin recombinase served as a better operator in vivo than the wild type site hixL (Hughes, K. T., Youderian, P., and Simon, M. I (1988) Genes & Dev. 2, 937-948). In vitro binding assays such as gel retardation assay and methylation protection assay demonstrated that Hin binds to hixC as tightly as it binds to hixL, even when the sites are located in negatively supercoiled plasmids. However, hixC served as a poor recombination site when it was subjected to the standard inversion assay in vitro. hixC showed a 16-fold slower inversion rate than the wild type. A series of biochemical assays designed to probe different stages of the Hin-mediated inversion reaction, demonstrated that Hin dimers bound to hixC have difficulty in forming paired hix site intermediates. KMnO4 and S1 nuclease assays detected an anomalous structure of the center of hixC only when the site was in negatively supercoiled plasmids. Mutational analysis in the central region of hixC and assays of paired hix site formation with topoisomers of the hixC substrate plasmid suggested that Hin is not able to pair hixC sites because of the presence of the anomalous structure in the center of the site. The structure does not behave like a DNA "cruciform" since Hin dimers still bind efficiently to the site. It is thought to consist of a short denatured "bubble" encompassing 2 base pairs. During the study of mutations in the center of hixC, it was found that Hin is not able to cleave DNA if a guanine residue is one of the two central nucleotides close to the cleavage site. Furthermore, Hin acts in a concerted fashion and cannot cleave any DNA strand if one of the four strands in the inversion intermediate is not cleavable.  相似文献   

13.
14.
The mechanism of the Cre recombinase of bacteriophage P1 in Escherichia coli cells was analyzed by topological methods in order to determine the important features of the in vivo reaction. Lambda infection was used to introduce the cre gene into cells containing plasmid substrates. The products of Cre resolution on substrates with directly repeated sites were predominantly free circles, even though decatenation by DNA gyrase was blocked by the drug norfloxacin. Recombination by Cre was greatly stimulated by negative supercoiling, and inversion occurred inefficiently. These results are strikingly different from those found with purified enzyme in vitro. Our data imply that Cre recombination in vivo is much more tightly controlled than it is in vitro, and that Cre acts predominantly as a resolvase in vivo. We suggest a role for Cre-mediated recombination in P1 plasmid amplification that is consistent with the selectivity of the enzyme in vivo.  相似文献   

15.
DNA inversions in phages and bacteria   总被引:3,自引:0,他引:3  
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.  相似文献   

16.
Efficient DNA inversion catalysed by the invertase Gin requires the cis-acting recombinational enhancer and the Escherichia coliFIS protein. Binding of FIS bends the enhancer DNA and, on a negatively supercoiled DNA inversion substrate, facilitates the formation of a synaptic complex with specific topology. Previous studies have indicated that FIS-independent Gin mutants can be isolated which have lost the topological constraints imposed on the inversion reaction yet remain sensitive to the stimulatory effect of FIS. Whether the effect of FIS is purely architectural, or whether in addition direct protein contacts between Gin and FIS are required for efficient catalysis has remained an unresolved question. Here we show that FIS mutants impaired in DNA binding are capable of either positively or negatively affecting the inversion reaction both in vivo and in vitro. We further demonstrate that the mutant protein FIS K25E/V66A/M67T dramatically enhances the cleavage of recombination sites by FIS-independent Gin in an enhancer-independent manner. Our observations suggest that FIS plays a dual role in the inversion reaction and stimulates both the assembly of the synaptic complex as well as DNA strand cleavage.  相似文献   

17.
CTnDOT encodes an integrase that is a member of the tyrosine recombinase family. The recombination reaction proceeds by sequential sets of genetic exchanges between the attDOT site in CTnDOT and an attB site in the chromosome. The exchanges are separated by 7 base pairs in each site. Unlike most tyrosine recombinases, IntDOT exchanges sites that contain different DNA sequences between the exchange sites to generate Holliday junctions (HJs) that contain mismatched bases. We demonstrate that IntDOT resolves synthetic HJs in vitro. Holliday junctions that contain identical sequences between the exchange sites are resolved into both substrates and products, while HJs that contain mismatches are resolved only to substrates. This result implies that resolution of HJs to products requires the formation of a higher-order nucleoprotein complex with natural sites containing IntDOT. We also found that proteins with substitutions of residues (V95, K94, and K96) in a putative alpha helix at the junction of the N and CB domains (coupler region) were defective in resolving HJs. Mutational analysis of charged residues in the coupler and the N terminus of the protein did not provide evidence for a charge interaction between the regions of the protein. V95 may participate in a hydrophobic interaction with another region of IntDOT.  相似文献   

18.
Cre recombinase is a prototypical member of the tyrosine recombinase family of site-specific recombinases. Members of this family of enzymes catalyze recombination between specific DNA sequences by cleaving and exchanging one pair of strands between the two substrate sites to form a 4-way Holliday junction (HJ) intermediate and then resolve the HJ intermediate to recombinant products by a second round of strand exchanges. Recently, hexapeptide inhibitors have been described that are capable of blocking the second strand exchange step in the tyrosine recombinase recombination pathway, leading to an accumulation of the HJ intermediate. These peptides are active in the lambda-integrase, Cre recombinase, and Flp recombinase systems and are potentially important tools for both in vitro mechanistic studies and as in vivo probes of cellular function. Here we present biochemical and crystallographic data that support a model where the peptide inhibitor binds in the center of the recombinase-bound DNA junction and interacts with solvent-exposed bases near the junction branch point. Peptide binding induces large conformational changes in the DNA strands of the HJ intermediate, which affect the active site geometries in the recombinase subunits.  相似文献   

19.
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.  相似文献   

20.
Site-specific recombination on supercoiled circular DNA molecules can yield a variety of knots and catenanes. Twist knots are some of the most common conformations of these products, and they can act as substrates for further rounds of site-specific recombination. They are also one of the simplest families of knots and catenanes. Yet, our systematic understanding of their implication in DNA and important cellular processes such as site-specific recombination is very limited. Here, we present a topological model of site-specific recombination characterizing all possible products of this reaction on twist knot substrates, extending the previous work of Buck and Flapan. We illustrate how to use our model to examine previously uncharacterized experimental data. We also show how our model can help determine the sequence of products in multiple rounds of processive recombination and distinguish between products of processive and distributive recombinations.This model studies generic site-specific recombination on arbitrary twist knot substrates, a subject for which there is limited global understanding. We also provide a systematic method of applying our model to a variety of different recombination systems.  相似文献   

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