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1.
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   

2.
A series of biochemical assays were developed and performed to monitor the molecular events that occur during the Hin-mediated DNA inversion reaction. These events can be divided into five different stages: 1) binding of proteins (Hin, Fis, and HU) to DNA; 2) pairing of Hin-binding sites; 3) invertasome formation; 4) DNA strand cleavage; 5) strand rotation and religation. A series of topoisomers of the wild type DNA substrate plasmid (ranging from fully relaxed molecules to those with more than the physiological superhelical density (the physiological superhelical density of pKH336 from Escherichia coli DH10B is -0.072 in this study)) was generated, and the role of negative supercoiling in each step of the inversion reaction was investigated. We found differences in the dependence of the formation of paired Hin-binding sites and of the invertasome formation on the superhelical density of the substrate plasmid. Pairing of Hin-binding sites occurs independently from invertasome formation, and a relatively low degree of negative supercoiling is enough to promote maximal pairing. However, efficient invertasome formation requires higher levels of negative supercoiling.  相似文献   

3.
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.
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   

6.
A previous genetic screen was designed to separate Hin recombinase mutants into distinct classes based on the stage in the recombination reaction at which they are blocked (O. Nanassy, Zoltan, and K. T. Hughes, Genetics 149:1649-1663, 1998). One class of DNA binding-proficient, recombination-deficient mutants was predicted by genetic classification to be defective in the step prior to invertasome formation. Based on the genetic criteria, mutants from this class were also inferred to be defective in interactions with Fis. In order to understand how the genetic classification relates to individual biochemical steps in the recombination reaction these mutants, R123Q, T124I, and A126T, were purified and characterized for DNA cleavage and recombination activities. Both the T124I and A126T mutants were partially active, whereas the R123Q mutant was inactive. The A126T mutant was not as defective for recombination as the T124I allele and could be partially rescued for recombination both in vivo and in vitro by increasing the concentration of Fis protein. Rescue of the A126T allele required the Fis protein to be DNA binding proficient. A model for a postsynaptic role for Fis in the inversion reaction is presented.  相似文献   

7.
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   

8.
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   

9.
Two critical steps controlling serine recombinase activity are the remodeling of dimers into the chemically active synaptic tetramer and the regulation of subunit rotation during DNA exchange. We identify a set of hydrophobic residues within the oligomerization helix that controls these steps by the Hin DNA invertase. Phe105 and Met109 insert into hydrophobic pockets within the catalytic domain of the same subunit to stabilize the inactive dimer conformation. These rotate out of the catalytic domain in the dimer and into the subunit rotation interface of the tetramer. About half of residue 105 and 109 substitutions gain the ability to generate stable synaptic tetramers and/or promote DNA chemistry without activation by the Fis/enhancer element. Phe106 replaces Phe105 in the catalytic domain pocket to stabilize the tetramer conformation. Significantly, many of the residue 105 and 109 substitutions support subunit rotation but impair ligation, implying a defect in rotational pausing at the tetrameric conformer poised for ligation. We propose that a ratchet-like surface involving Phe105, Met109 and Leu112 within the rotation interface functions to gate the subunit rotation reaction. Hydrophobic residues are present in analogous positions in other serine recombinases and likely perform similar functions.  相似文献   

10.
Lee HJ  Lee YL  Ji JJ  Lim HM 《Molecules and cells》2003,16(3):377-384
The biochemical reaction of a site-specific recombinase such as Hin invertase or gammadelta resolvase starts with binding of the recombinase to its recombination site and cleavage of the DNA in the center of the site. This is followed by strand exchange and finally ligation of the ends of the recombined strands. Previous biochemical studies have shown that Hin invertase and gammadelta resolvase cannot proceed beyond DNA cleavage in the absence of Mg++ ion, indicating that these recombinases require Mg++ ion in the strand exchange process. We have observed that the intercalating agent, ethidium bromide (2 microM), does not interfere with DNA cleavage, but slows strand exchange in a concentration-dependent manner. Levels of Mg++ ion below 5 mM also slow strand exchange substantially. We infer that random intercalation of ethidium bromide inhibits unwinding of the double helix at the recombination site in the negatively supercoiled DNA and propose that Mg+ may be required for Hin to deform the secondary structure of B-DNA prior to strand exchange.  相似文献   

11.
3-Isopropylmalate dehydrogenase (IPMDH) is a dimeric enzyme with a strongly hydrophobic core that is composed of residues from four alpha-helices. We replaced Glu253, which is found in the hydrophobic core and is part of the subunit interface of the Bacillus subtilis (Bs) IPMDH, with several other amino acids to probe. The thermostabilities of the mutants were assessed by measuring the residual enzymatic activities at 40 degrees C after heat treatment and by monitoring changes in ellipticity at 222 nm as the environmental temperature increased incrementally. The results of these studies indicate that, for residues with non-polar side chains, when positioned at residue 253, the thermostabilities of their corresponding mutants correlate positively with the relative hydrophobicities of the side chains. Relative activities of all mutants are lower than that of the wild-type enzyme. For two of the mutants, we directly show that the substitution at position 253 negatively affects Mn(2+) binding, which is required for catalysis. When a lysine is the position 253 residue, the protein dissociates. The results presented herein increase our understanding of the role played by the BsIPMDH dimer interface on the stability and activity of BsIPMDH.  相似文献   

12.
A series of recombinational enhancer mutants was constructed by manipulating the ClaI site between the two FIS binding sites of the Hin enhancer. These mutants include insertions from two to 12 base-pairs and two deletions of one or two base-pairs. Recombinational enhancer activity was found only with four mutants carrying either a four base-pair substitution, ten base-pair insertions or a one base-pair deletion, respectively; two other ten base-pair insertion mutants, however, were inactive, although FIS protein binding was unaffected. So, besides binding of FIS protein to its specific sites within the enhancer sequence and the correct helical positioning of these sites on the DNA, another criterion for enhancer activity must be fulfilled. DNA bending assays identify this requirement as a change of the enhancer DNA conformation, which FIS protein is able to induce and to stabilize. This conformational change of the DNA can be blocked by mutations in the central segment between the two FIS binding sites of the Hin enhancer. This sequence has special functions for the recombinational enhancer activity.  相似文献   

13.
The lactose transport protein (LacS) of Streptococcus thermophilus belongs to a family of transporters in which putative alpha-helices II and IV have been implicated in cation binding and the coupled transport of the substrate and the cation. Here, the analysis of site-directed mutants shows that a positive and negative charge at positions 64 and 71 in helix II are essential for transport, but not for lactose binding. The conservation of charge/side-chain properties is less critical for Glu-67 and Ile-70 in helix II, and Asp-133 and Lys-139 in helix IV, but these residues are important for the coupled transport of lactose together with a proton. The analysis of second-site suppressor mutants indicates an ion pair exists between helices II and IV, and thus a close approximation of these helices can be made. The second-site suppressor analysis also suggests ion pairing between helix II and the intracellular loops 6-7 and 10-11. Because the C-terminal region of the transmembrane domain, especially helix XI and loop 10-11, is important for substrate binding in this family of proteins, we propose that sugar and proton binding and translocation are performed by the joint action of these regions in the protein. Indeed, substrate protection of maleimide labeling of single cysteine mutants confirms that alpha-helices II and IV are directly interacting or at least conformationally involved in sugar binding and/or translocation. On the basis of new and published data, we reason that the helices II, IV, VII, X, and XI and the intracellular loops 6-7 and 10-11 are in close proximity and form the binding sites and/or the translocation pathway in the transporters of the galactosides-pentosides-hexuronides family.  相似文献   

14.
TraR is a LuxR-type quorum-sensing protein encoded by the tumour-inducing plasmid of Agrobacterium tumefaciens . TraR requires the pheromone N-3-oxooctanoyl- l -homoserine lactone (OOHL) for biological activity, and is dimeric both in solution and when bound to DNA. Dimerization is mediated primarily by two α-helices, one in the N-terminal OOHL binding domain, and the other in the C-terminal DNA binding domain. Each of these helices forms a parallel coiled coil with the identical helix of the opposite subunit. We have previously shown that OOHL is essential for resistance to proteolysis, and here we asked whether dimerization is also required for protease resistance. We constructed a series of site-directed mutations at the dimer interface, and tested these mutants for activity in vivo . Alteration of residues A149, A150, A153, A222 and I229 completely abolished activity, while alteration of three other residues also caused significant defects. All mutants were tested for dimerization as well as for specific DNA binding. The cellular abundance of these proteins in A. tumefaciens was measured using Western immunoblots and OOHL sequestration, while the half-life was measured by pulse-chase radiolabelling. We found a correlation between defects in in vivo activity, in vitro dimerization, DNA binding and protein half-life. We conclude that dimerization of TraR enhances resistance to cellular proteases.  相似文献   

15.
Incorporation of the DNA-cleaving moiety EDTA.Fe at discrete amino acid residues along a DNA-binding protein allows the positions of these residues relative to DNA bases, and hence the organization of the folded protein, to be mapped by high-resolution gel electrophoresis. A 52-residue protein, based on the sequence-specific DNA-binding domain of Hin recombinase (139-190), with EDTA at the NH2 terminus cleaves DNA at Hin recombination sites. The cleavage data for EDTA-Hin(139-190) reveal that the NH2 terminus of Hin(139-190) is bound in the minor groove of DNA near the symmetry axis of Hin-binding sites [Sluka, J. P., Horvath, S. J., Bruist, M. F., Simon, M. I., & Dervan, P. B. (1987) Science 238, 1129]. Six proteins, varying in length from 49 to 60 residues and corresponding to the DNA-binding domain of Hin recombinase, were synthesized by solid-phase methods: Hin(142-190), Hin(141-190), Hin(140-190), Hin(139-190), Hin(135-190), and Hin(131-190) were prepared with and without EDTA at the NH2 termini in order to test the relative importance of the residues Gly139-Arg140-Pro141-Arg142, located near the minor groove, for sequence-specific recognition at five imperfectly conserved 12-base-pair binding sites. Footprinting and affinity cleaving reveal that deletion of Gly139 results in a protein with affinity and specificity similar to those of Hin(139-190) but that deletion of Gly139-Arg140 affords a protein with altered affinities and sequence specificities for the five binding sites. It appears that Arg140 in the DNA-binding domain of Hin is important for recognition of the 5'-AAA-3' sequence in the minor groove of DNA. Our results indicate modular DNA and protein interactions with two adjacent DNA sites (major and minor grooves, respectively) bound on the same face of the helix by two separate parts of the protein.  相似文献   

16.
Bacteria evade the effects of cytotoxic compounds through the efflux activity of membrane-bound transporters such as the small multidrug resistance (SMR) proteins. Consisting typically of ca. 110 residues with four transmembrane (TM) α-helices, crystallographic studies have shown that TM helix 1 (TM1) through TM helix 3 (TM3) of each monomer create a substrate binding "pocket" within the membrane bilayer, while a TM4-TM4 interaction accounts for the primary dimer formation. Previous work from our lab has characterized a highly conserved small-residue heptad motif in the Halobacterium salinarum transporter Hsmr as (90)GLXLIXXGV(98) that lies along the TM4-TM4 dimer interface of SMR proteins as required for function. Focusing on conserved positions 91, 93, 94, and 98, we substituted the naturally occurring Hsmr residue for Ala, Phe, Ile, Leu, Met, and Val at each position in the Hsmr TM4-TM4 interface. Large-residue replacements were studied for their ability to dimerize on SDS-polyacrylamide gels, to bind the cytotoxic compound ethidium bromide, and to confer resistance by efflux. Although the relative activity of mutants did not correlate with dimer strength for all mutants, all functional mutants lay within 10% of dimerization relative to the wild type (WT), suggesting that the optimal dimer strength at TM4 is required for proper efflux. Furthermore, nonfunctional substitutions at the center of the dimerization interface that do not alter dimer strength suggest a dynamic TM4-TM4 "pivot point" that responds to the efflux requirements of different substrates. This functionally critical region represents a potential target for inhibiting the ability of bacteria to evade the effects of cytotoxic compounds.  相似文献   

17.
Eight Tn10 Tet repressor mutants with an induction-deficient phenotype and with primary mutations located at or close to the dimer interface were mutagenized and screened for inducibility in the presence of tetracycline. The second-site suppressors with wild-type-like operator binding activity that were obtained act, except for one, at a distance, suggesting that they contribute to conformational changes in the Tet repressor. Many of these long-range suppressors occur along the dimer interface, indicating that interactions between the monomers play an important role in Tet repressor induction.  相似文献   

18.
The high-resolution structure of the 43 kDa N-terminal fragment of the DNA gyrase B protein shows a large cavity within the protein dimer. The approximate size of this cavity is 20 A, suggesting it could accommodate a DNA helix. Computer-modelling studies of this cavity suggest that it contains a constriction, reducing the width to approximately 13 A, principally caused by the side chain of Arg286. We have used site-directed mutagenesis to alter this residue to Gln. Gyrase bearing this mutation shows virtually no supercoiling activity and near-normal relaxation and DNA cleavage activities. The mutated protein has ATPase activity which cannot be stimulated by DNA. These data support the proposed role of the 43 kDa domain as an ATP-operated clamp which binds DNA during the supercoiling cycle. The lack of DNA-dependent ATPase of the mutant may indicate that binding of DNA within the clamp is a prerequisite for stimulation of the ATPase activity.  相似文献   

19.
20.
The tetrameric paramyxovirus hemagglutinin-neuraminidase (HN) protein mediates attachment to sialic acid-containing receptors as well as cleavage of the same moiety via its neuraminidase (NA) activity. The X-ray crystallographic structure of an HN dimer from Newcastle disease virus (NDV) suggests that a single site in two different conformations mediates both of these activities. This conformational change is predicted to involve an alteration in the association between monomers in each HN dimer and to be part of a series of changes in the structure of HN that link its recognition of receptors to the activation of the other viral surface glycoprotein, the fusion protein. To explore the importance of the dimer interface to HN function, we performed a site-directed mutational analysis of residues in a domain defined by residues 218 to 226 at the most membrane-proximal part of the dimer interface in the globular head. Proteins carrying substitutions for residues F220, S222, and L224 in this domain were fusion deficient. However, this fusion deficiency was not due to a direct effect of the mutations on fusion. Rather, the fusion defect was due to a severely impaired ability to mediate receptor recognition at 37 degrees C, a phenotype that is not attributable to a change in NA activity. Since each of these mutated proteins efficiently mediated attachment in the cold, it was also not due to an inherent inability of the mutated proteins to recognize receptors. Instead, the interface mutations acted by weakening the interaction between HN and its receptor(s). The phenotype of these mutants correlates with the disruption of intermonomer subunit interactions.  相似文献   

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