Determinants of correct res site alignment in site-specific recombination by Tn3 resolvase.

Bednarz, Amy L.; Boocock, Martin R.; Sherratt, David J.

doi:10.1101/gad.4.12b.2366

Cited by 51 publications

(40 citation statements)

References 37 publications

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“…In other reaction conditions, we also observed intermolecular recombination (data not shown). These results conform with previous studies of resolvase-mediated recombination at site I (15,18). The extent of recombination in vitro was less than that observed in E. coli, for reasons that are as yet unclear.…”

Section: Resultssupporting

confidence: 82%

Chimeric recombinases with designed DNA sequence recognition

Akopian

Boocock

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

107

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Site-specific recombination typically occurs only between DNA sequences that have co-evolved with a natural recombinase enzyme to optimize sequence recognition, catalytic efficiency, and regulation. Here, we show that the sequence recognition and the catalysis functions of a recombinase can be specified by unrelated protein domains. We describe chimeric recombinases with a catalytic domain from an activated multiple mutant of the bacterial enzyme Tn3 resolvase, fused to a DNA recognition domain from the mouse transcription factor Zif268. These proteins catalyze efficient recombination specifically at synthetic target sites recognized by two Zif268 domains. Our results demonstrate the functional autonomy of the resolvase catalytic domain and open the way to creating ''custom-built'' recombinases that act at chosen natural target sequences.

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Section: Resultssupporting

confidence: 82%

Chimeric recombinases with designed DNA sequence recognition

Akopian

Boocock

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

107

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“…3A). The terms 'parallel' and 'antiparallel' have been used previously to describe alternative versions of the synapse (Bednarz et al, 1990;Moskowitz et al, 1991). It should be noted that we do not use these terms to infer the actual paths of the DNA within the synaptic complex.…”

Section: Tamentioning

confidence: 99%

“…Mutation of the proposed catalytic serine residue in f C31 integrase abolishes recombination function suggesting that this protein catalyses recombination using a similar mechanism to gd and Tn 3 resolvases (Thorpe and Smith, 1998). The first step in gd and Tn 3 resolution is the formation of a productive synapse in which resolvase binds to sequences in two identical res sites, bringing the two DNA strands containing the crossover sites together in a specific complex (Bednarz et al ., 1990;Sarkis et al ., 2001;Grindley, 2002). A considerable body of evidence suggests that strand exchange occurs via the double-strand cut rotation model (Grindley, 2002).…”

Section: Introductionmentioning

confidence: 99%

Switching the polarity of a bacteriophage integration system

Smith

Till

Smith

2004

Molecular Microbiology

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SummaryDuring lysogenic growth many temperate bacteriophage genomes are integrated into the host's chromosome and efficient integration and excision are therefore an essential part of the phage life cycle. The Streptomyces phage f f f f C31 encodes an integrase related to the resolvase/invertases and is evolutionarily and mechanistically distinct from the integrase of phage l l l l . We show that during f f f f C31 integration the polarity of the recombination sites, attB and attP , is dependent on the sequences of the two base pairs (bp) where crossover occurs. A loss or switch in polarity of the recombination sites can occur by mutation of this dinucleotide, leading to incorrectly joined products. The properties of the mutant sites implies that f f f f C31 integrase interacts symmetrically with the substrates, which during synapsis can align apparently freely in either of two alternative forms that lead to correct or incorrect joining of products. Analysis of the topologies of the reaction products provided evidence that integrase can synapse and activate strand exchange even when recombinant products cannot form due to mismatches at the crossover site. The topologies of the recombination products are complex and indicative of multiple pathways to product formation. The efficiency of integration of a f f f f C31 derivative, KC859, into an attB site with switched polarity was assayed in vivo and shown to be no different from integration into a wild-type attB . Thus neither the host nor KC859 express a factor that influences the alignment of the recombination sites at synapsis.

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“…These additional binding sites have been shown to dictate the strict specificity of resolvase-mediated recombination (Dr6ge and Cozzarelli 1989;Stark et al 1989). Thus, like the Hin system, synapsis of the res recombination sites is not determined exclusively by a particular parallel or antiparallel orientation (Bednarz et al 1990). Rather, alignment is dependent on the preferential formation of a particular synaptic intermediate, which leads to the bias of one recombinant product over the other.…”

Section: Determinants Of Recombination Site Alignmentmentioning

confidence: 99%

Alignment of recombination sites in Hin-mediated site-specific DNA recombination.

Moskowitz¹,

Heichman²,

Johnson³

1991

Genes Dev.

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The Hin site-specific recombination system normally promotes inversion of DNA between two recombination sites in inverted orientation. We show that the rate of deletion of DNA between two directly repeated recombination sites is 10-300 times slower than inversion between sites in their native configuration as measured in vivo and in vitro, respectively. In vitro studies have shown that the deletion reaction has the same requirement for Fis, a recombinational enhancer, and DNA supercoiling as the inversion reaction. These requirements, together with the finding that the deletion products are interlinked once suggest that the deletion synaptic complex is similar to the invertasome intermediate that generates inversion. The inefficiency of the deletion reaction is not a function of a reduced ability to recognize or synapse recombination sites in direct orientation. Not only do these substrates support an efficient knotting reaction, but directly repeated recombination sites with symmetric core sequences also invert efficiently. These findings demonstrate that the recombination sites are preferentially assembled into the invertasome structure with the sites aligned in the configuration for inversion regardless of their starting orientation. We propose that the dynamics of a supercoiled DNA molecule biases the geometric assembly of specific intermediates. In the case of Hin-mediated recombination, inversion is overwhelmingly preferred over deletion because DNA supercoiling favors a specific alignment of DNA strands in the synaptic complex.[Key Words: Hin; Fis; recombinational enhancer; site-specific recombination; DNA strand exchange; DNA supercoiling] Received March 15, 1991; revised version accepted June 18, 1991.The product of Hin-mediated site-specific recombination is the inversion of a 995-bp DNA segment in the Salmonella chromosome. Inversion of DNA between the two recombination sites switches the orientation of the promoter that transcribes the H2 flagellin gene and the repressor of the unlinked H1 flagellin gene, resulting in the alternate expression of the H1 and H2 flagellins (Silverman and Simon 1980;Zieg and Simon 1980). Scott and Simon (1982) demonstrated that Hin-mediated recombination in vivo resulted in deletion of the intervening DNA when the orientation of one of the recombination sites is reversed on a plasmid substrate. However, deletion of DNA located between the directly repeated recombination sites occurred much less efficiently than inversion between recombination sites in their native orientation. The related Gin and Cin recombinases invert a segment of DNA containing tail fiber genes in phage Mu and P1, respectively (Kamp et al. 1978;Iida et al. 1982). In each of these systems, the inversion reaction 2present address:

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Determinants of correct res site alignment in site-specific recombination by Tn3 resolvase.

Cited by 51 publications

References 37 publications

Chimeric recombinases with designed DNA sequence recognition

Chimeric recombinases with designed DNA sequence recognition

Switching the polarity of a bacteriophage integration system

Alignment of recombination sites in Hin-mediated site-specific DNA recombination.

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