Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.2.1.26 (invertase)
4,927 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Site-specific recombination requires conserved DNA sequences specific to each system, and system-specific proteins that recognize specific DNA sequences. The site-specific recombinases seem to fall into at least two families, based on their protein structure and chemistry of strand breakage. One of these is the resolvase-invertase family, members of which seem to form a serine-phosphate linkage with DNA. Members of the other family, called the integrase family, contain a conserved tyrosine residue that forms a covalent linkage with the 3'-phosphate of DNA at the site of recombination. Structural comparison of integrases shows that these proteins share a highly conserved 40-residue motif. V-(D)-J recombination of the immunoglobulin gene requires conserved recombination signal sequences (RS) of a heptamer CACTGTG and a T-rich nonamer GGTTTTTGT, which are separated by a spacer sequence of either 12 or 23 bases We have recently purified, almost to homogeneity, a protein that specifically binds to the immunoglobulin J kappa RS containing the 23-base-pair spacer sequence. By synthesizing probes on the basis of partial amino-acid sequences of the purified protein, we have now isolated and characterized the complementary DNA of this protein. The amino-acid sequence deduced from the cDNA sequence reveals that the J kappa RS-binding protein has a sequence similar to the 40-residue motif of integrases of phages, bacteria and yeast, indicating that this protein could be involved in V-(D)-J recombination as a recombinase.
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PMID:A protein binding to the J kappa recombination sequence of immunoglobulin genes contains a sequence related to the integrase motif. 255 44

The chloramphenicol-resistance transposon Tn4451 undergoes precise conjugative deletion from its parent plasmid plP401 in Clostridium perfringens and precise spontaneous excision from multicopy plasmids in Escherichia coli. The complete nucleotide sequence of the 6338 bp transposon was determined and it was found to encode six genes. Genetic analysis demonstrated that the largest Tn4451-encoded gene, tnpX, was required for the spontaneous excision of the transposon in both E. coli and C. perfringens, since a Tn4451 derivative that lacked a functional tnpX gene was completely stable in both organisms. Because the ability of this derivative to excise was restored by providing the tnpX gene on a compatible plasmid, it was concluded that this gene encoded a trans-acting site-specific recombinase. Allelic exchange was used to introduce the tnpX delta 1 allele onto plP401 and it was shown that TnpX was also required for the conjugative excision of Tn4451 in C. perfringens. It was also shown by hybridization and polymerase chain reaction (PCR) studies that TnpX-mediated transposon excision resulted in the formation of a circular form of the transposon. The TnpX recombinase was unique because it potentially contained the motifs of two independent site-specific recombinase families, namely the resolvase/invertase and integrase families. Sequence analysis indicated that the resolvase/invertase domain of TnpX was likely to be involved in the excision process by catalysing the formation of a 2 bp staggered nick on either side of the GA dinucleotide located at the ends of the transposon and at the junction of the circular form. The other Tn4451-encoded genes include tnpZ, which appears to encode a second potential site-specific recombinase. This protein has similarity to plasmid-encoded Mob/Pre proteins, which are involved in plasmid mobilization and multimer formation. Located upstream of the tnpZ gene was a region with similarity to the site of interaction of these mobilization proteins.
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PMID:Molecular genetics of the chloramphenicol-resistance transposon Tn4451 from Clostridium perfringens: the TnpX site-specific recombinase excises a circular transposon molecule. 756 13

Integrons confer on bacterial plasmids a capability of taking up antibiotic resistance genes by integrase-mediated recombination. We show here that integrons are situated on genetic elements flanked by 25-bp inverted repeats. The element carrying the integron of R751 has three segments conserved with similar elements in Tn21 and Tn5086. Several characteristics suggest that this element is a transposon, which we call Tn5090. Tn5090 was shown to contain an operon with three open reading frames, of which two, tniA and tniB, were predicted by amino acid similarity to code for transposition proteins. The product of tniA (559 amino acids) is a probable transposase with 25% amino acid sequence identity to TnsB from Tn7. Both of these polypeptides contain the D,D(35)E motif characteristic of a protein family made up of the retroviral and retrotransposon IN proteins and some bacterial transposases, such as those of Tn552 and of a range of insertion sequences. Like the transposase genes in Tn552, Mu, and Tn7, the tniA gene was followed by a gene, tniB, for a probable ATP-binding protein. The ends of Tn5090, like those of most other elements producing D,D(35)E proteins, begin by 5'-TG and also contains a complex structure with four 19-bp repeats at the left end and three at the right end. Similarly organized repeats have been observed earlier at the termini of both Tn7 and phage Mu, where they bind their respective transposases and have a role in holoenzyme assembly. Another open reading frame observed in Tn5090, tniC, codes for a recombinase of the invertase/resolvase family, suggesting a replicative transposition mechanism. The data presented here suggest that Tn5090, Tn7, Tn552, and Mu form a subfamily of bacterial transposons which in parallel to many insertion sequences are related to the retroelements.
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PMID:Transposon Tn5090 of plasmid R751, which carries an integron, is related to Tn7, Mu, and the retroelements. 819 81

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.
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PMID: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. 926 Sep 58

The genome of the broad host range Streptomyces temperate phage, phiC31, is known to integrate into the host chromosome via an enzyme that is a member of the resolvase/invertase family of site-specific recombinases. The recombination properties of this novel integrase on the phage and Streptomyces ambofaciens attachment sites, attP and attB, respectively, were investigated in the heterologous host, Escherichia coli, and in an in vitro assay by using purified integrase. The products of attP/B recombination, i.e., attL and attR, were identical to those obtained after integration of the prophage in S. ambofaciens. In the in vitro assay only buffer, purified integrase, and DNAs encoding attP and attB were required. Recombination occurred irrespective of whether the substrates were supercoiled or linear. A mutant integrase containing an S12F mutation was completely defective in recombination both in E. coli and in vitro. No recombination was observed between attB/attB, attP/attP, attL/R, or any combination of attB or attP with attL or attR, suggesting that excision of the prophage (attL/R recombination) requires an additional phage- or Streptomyces-encoded factor. Recombination could occur intramolecularly to cause deletion between appropriately orientated attP and attB sites. The results show that directionality in phiC31 integrase is strictly controlled by nonidentical recombination sites with no requirement to form the topologically defined structures that are more typical of the resolvases/invertases.
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PMID:In vitro site-specific integration of bacteriophage DNA catalyzed by a recombinase of the resolvase/invertase family. 957 12

The haloalkane-degrading bacteria Rhodococcus rhodochrous NCIMB13064, Pseudomonas pavonaceae 170, and Mycobacterium sp. strain GP1 share a highly conserved haloalkane dehalogenase gene (dhaA). Here, we describe the extent of the conserved dhaA segments in these three phylogenetically distinct bacteria and an analysis of their flanking sequences. The dhaA gene of the 1-chlorobutane-degrading strain NCIMB13064 was found to reside within a 1-chlorobutane catabolic gene cluster, which also encodes a putative invertase (invA), a regulatory protein (dhaR), an alcohol dehydrogenase (adhA), and an aldehyde dehydrogenase (aldA). The latter two enzymes may catalyze the oxidative conversion of n-butanol, the hydrolytic product of 1-chlorobutane, to n-butyric acid, a growth substrate for many bacteria. The activity of the dhaR gene product was analyzed in Pseudomonas sp. strain GJ1, in which it appeared to function as a repressor of dhaA expression. The 1,2-dibromoethane-degrading strain GP1 contained a conserved DNA segment of 2.7 kb, which included dhaR, dhaA, and part of invA. A 12-nucleotide deletion in dhaR led to constitutive expression of dhaA in strain GP1, in contrast to the inducible expression of dhaA in strain NCIMB13064. The 1, 3-dichloropropene-degrading strain 170 possessed a conserved DNA segment of 1.3 kb harboring little more than the coding region of the dhaA gene. In strains 170 and GP1, a putative integrase gene was found next to the conserved dhaA segment, which suggests that integration events were responsible for the acquisition of these DNA segments. The data indicate that horizontal gene transfer and integrase-dependent gene acquisition were the key mechanisms for the evolution of catabolic pathways for the man-made chemicals 1, 3-dichloropropene and 1,2-dibromoethane.
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PMID:Roles of horizontal gene transfer and gene integration in evolution of 1,3-dichloropropene- and 1,2-dibromoethane-degradative pathways. 1073 62

The construction of a delivery and clearing system for the generation of food-grade recombinant lactic acid bacterium strains, based on the use of an integrase (Int) and a resolvo-invertase (beta-recombinase) and their respective target sites (attP-attB and six, respectively) is reported. The delivery system contains a heterologous replication origin and antibiotic resistance markers surrounded by two directly oriented six sites, a multiple cloning site where passenger DNA could be inserted (e.g., the cI gene of bacteriophage A2), the int gene, and the attP site of phage A2. The clearing system provides a plasmid-borne gene encoding beta-recombinase. The nonreplicative vector-borne delivery system was transformed into Lactobacillus casei ATCC 393 and, by site-specific recombination, integrated as a single copy in an orientation- and Int-dependent manner into the attB site present in the genome of the host strain. The transfer of the clearing system into this strain, with the subsequent expression of the beta-recombinase, led to site-specific DNA resolution of the non-food-grade DNA. These methods were validated by the construction of a stable food-grade L. casei ATCC 393-derived strain completely immune to phage A2 infection during milk fermentation.
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PMID:Generation of food-grade recombinant lactic acid bacterium strains by site-specific recombination. 1083 43

The genome of the Streptomyces temperate phage phiC31 integrates into the host chromosome via a recombinase belonging to a novel group of phage integrases related to the resolvase/invertase enzymes. Previously, it was demonstrated that, in an in vitro recombination assay, phiC31 integrase catalyses integration (attP/attB recombination) but not excision (attL/attR). The mechanism responsible for this recombination site selectivity was therefore investigated. Purified integrase was shown to bind with similar apparent binding affinities to between 46 bp and 54 bp of DNA at each of the attachment sites, attP, attB, attL and attR. Assays using recombination sites of 50 bp and 51 bp for attP and attB, respectively, showed that these fragments were functional in attP/attB recombination and maintained strict site selectivity, i.e. no recombination between non-permissive sites, such as attP/attP, attB/attL, etc., was observed. Using bandshifts and supershift assays in which permissive and non-permissive combinations of att sites were used in the presence of integrase, only the attP/attB combination could generate supershifts. Recombination products were isolated from the supershifted complexes. It was concluded that these supershifted complexes contained the recombination synapse and that site specificity, and therefore directionality, is determined at the level of stable synapse formation.
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PMID:Control of directionality in the site-specific recombination system of the Streptomyces phage phiC31. 1106 50

Piv, a site-specific invertase from Moraxella lacunata, exhibits amino acid homology with the transposases of the IS110/IS492 family of insertion elements. The functions of conserved amino acid motifs that define this novel family of both transposases and site-specific recombinases (Piv/MooV family) were examined by mutagenesis of fully conserved amino acids within each motif in Piv. All Piv mutants altered in conserved residues were defective for in vivo inversion of the M. lacunata invertible DNA segment, but competent for in vivo binding to Piv DNA recognition sequences. Although the primary amino acid sequences of the Piv/MooV recombinases do not contain a conserved DDE motif, which defines the retroviral integrase/transposase (IN/Tnps) family, the predicted secondary structural elements of Piv align well with those of the IN/Tnps for which crystal structures have been determined. Molecular modelling of Piv based on these alignments predicts that E59, conserved as either E or D in the Piv/MooV family, forms a catalytic pocket with the conserved D9 and D101 residues. Analysis of Piv E59G confirms a role for E59 in catalysis of inversion. These results suggest that Piv and the related IS110/IS492 transposases mediate DNA recombination by a common mechanism involving a catalytic DED or DDD motif.
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PMID:Conserved amino acid motifs from the novel Piv/MooV family of transposases and site-specific recombinases are required for catalysis of DNA inversion by Piv. 1116 5

The site-specific recombination system of temperate lactococcal bacteriophage TP901-1 is unusual in several respects. First, the integrase belongs to the family of extended resolvases rather than to the lambda integrase family and second, in the presence of this integrase, a 56 bp attP fragment is sufficient for efficient recombination with the chromosomal attB site in the host Lactococcus lactis subsp. cremoris MG1363. In the present work, this attB site was analysed and a 43 bp attB region was found to be the smallest fragment able to participate fully in recombination. In vitro studies showed that the TP901-1 integrase binds this 43 bp attB fragment, the 56 bp attP and a larger attP fragment with equal affinity. Mutational analysis of the 5 bp common core region (TCAAT) showed that the TC dinucleotide is essential for recombination, but not for binding of the integrase, whereas none of the last three bases are important for recombination. When a number of attL sites, obtained by recombination between an attB site containing a mutation in this TC dinucleotide and a wild-type attP site, were sequenced, a mix of sites with the wild-type or the mutated sequence was obtained. These results are consistent with the hypothesis that the TC dinucleotide constitutes the TP901-1 overlap region. A 2 bp overlap region has been observed in recombination reactions catalysed by all other members of the resolvase/invertase family tested so far. By selecting for attB sites with a decreased ability to participate in recombination, two bases located outside the core region of attB were shown to be involved in the in vitro binding of the TP901-1 integrase.
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PMID:Resolvase-like recombination performed by the TP901-1 integrase. 1149 84


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