Site-specific recombination in bacteriophage Mu: characterization of binding sites for the DNA invertase Gin.

Mertens, Gabriele; Klippel, Anke; Fuss, Heidemarie; Blöcker, Helmut; Frank, Ronald; Kahmann, Regine

doi:10.1002/j.1460-2075.1988.tb02934.x

Cited by 41 publications

(32 citation statements)

References 30 publications

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“…TC\GA overlap regions have been described both for the Gin invertase of bacteriophage Mu (Mertens et al, 1988) and for the extended resolvase TnpX, which is involved in site-specific excision of the conjugative transposon Tn4451 (Crellin & Rood, 1997 ;Bannam et al, 1995). A mutation analysis revealed that changing the T of the Tn4451 overlap region has a more profound effect on recombination than changing the C, in agreement with the observations in the present study.…”

Section: The Tp901-1 Overlap Regionsupporting

confidence: 91%

Resolvase-like recombination performed by the TP901-1 integrase The GenBank accession number for the sequence reported in this paper is Y15043.

2001

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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 λ 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 wildtype 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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Section: The Tp901-1 Overlap Regionsupporting

confidence: 91%

Resolvase-like recombination performed by the TP901-1 integrase The GenBank accession number for the sequence reported in this paper is Y15043.

2001

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“…The data suggest that TnpX, in addition to recognizing the ends of the transposon during Tn4451 excision, also has a role in the insertion of the transposon at the target site. Finally, both the target sites and the circular form junction also resembled the gix and bix sites which are recognized by the Gin and PinB invertases from phage Mu and Shigella boydii, respectively (28,47). These invertases introduce staggered 2-bp cuts at the GA dinucleotides during the inversion reactions.…”

Section: Resultsmentioning

confidence: 96%

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

Crellin

Rood

1997

J Bacteriol

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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. The 6.3-kb transposable element Tn4451 is encoded on the conjugative R plasmid pIP401 from the anaerobic pathogen Clostridium perfringens and encodes resistance to chloramphenicol. The transposon undergoes precise conjugative excision following the transfer of pIP401 in C. perfringens and precise spontaneous excision from multicopy plasmids in both C. perfringens and Escherichia coli (2, 4, 9). A second, closely related element, Tn4452, has also been identified and behaves in a manner similar to Tn4451. Transposition of both elements has been detected in E. coli at very low frequencies (2), but transposition has not been detected in the native host.The entire nucleotide sequence of Tn4451 has been determined (9). The element has six genes, one of which, tnpX, has been shown to encode a trans-acting site-specific recombinase that is responsible for both the spontaneous and conjugative excision of Tn4451. Transposon derivatives with a tnpX gene, designated tnpX⌬1, that contains an internal deletion are stable on multicopy plasmids in C. perfringens and E. coli, and the introduction of the tnpX⌬1 allele into pIP401 results in a Tn4451 derivative that is stable upon conjugation. The TnpX protein catalyzes the excision of Tn4451 as a circular molecule which, by analogy with the well-characterized conjugative transposons Tn916 and Tn1545 (40), may well be the transposition intermediate. Tn4451 is flanked by directly repeated GA dinucleotides (3). Following the excision of Tn4451 (see Fig. 1), a GA dinucleotide is found at the junction of the circular form, where th...

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“…In these plasmids, the fis gene is cloned under the control of an IPTG-inducible promoter. The plasmid pAK3 is a pBR derivative containing the Mu recombinational enhancer with three specific FIS-binding sites (Mertens et al, 1988). The pSC101 plasmid, which carries a tetracycline resistance gene, was kindly provided by W. Messer.…”

Section: Plasmids and Constructsmentioning

confidence: 99%

FIS modulates growth phase‐dependent topological transitions of DNA in Escherichia coli

Schneider

Travers

Muskhelishvili

1997

Molecular Microbiology

117

104

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SummaryThe Escherichia coli DNA-binding protein FIS serves as a DNA architectural factor in two unrelated enzymatic reactions, the site-specific inversion of DNA and transcriptional activation of stable RNA promoters. In both these processes, FIS facilitates the assembly and dynamic transitions of two structurally distinct nucleoprotein complexes. We have proposed previously that, in these systems, FIS stabilizes writhed DNA microloops by binding at multiple helically phased sites in DNA. However, FIS also binds and bends DNA at many non-specific sites and, at its maximum levels in the early exponential phase, FIS could potentially occupy a considerable part of the E. coli chromosome. Here, we show that fis affects growth phase-specific alterations in the supercoiling level of DNA. Expression of fis accelerates the accumulation of moderately supercoiled plasmids in stationary phase, which are stabilized by FIS after nutritional shift-up. In accordance with such a function, FIS modulates the relaxing and supercoiling activities of topoisomerases in vitro in a way that keeps DNA in a moderately supercoiled state. Our results suggest that the primary role of FIS is to modulate chromosomal dynamics during bacterial growth.

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Site-specific recombination in bacteriophage Mu: characterization of binding sites for the DNA invertase Gin.

Cited by 41 publications

References 30 publications

Resolvase-like recombination performed by the TP901-1 integrase The GenBank accession number for the sequence reported in this paper is Y15043.

Resolvase-like recombination performed by the TP901-1 integrase The GenBank accession number for the sequence reported in this paper is Y15043.

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

FIS modulates growth phase‐dependent topological transitions of DNA in Escherichia coli

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