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A new in vitro strand transfer assay for monitoring bacterial class 1 integron recombinase IntI1 activity.

Dubois V, Debreyer C, Litvak S, Quentin C, Parissi V - PLoS ONE (2007)

Bottom Line: Results show that IntI1 possesses all the features needed for performing recombination between attI and attC sites.However, differences in the in vitro intermolecular recombination efficiencies were found according to the target sites and were correlated with DNA affinities of the enzyme but not with in vivo data.The differential affinity of the enzyme for each site, its capacity to bind to a single-stranded structure at the attC site and the recombination observed with single-stranded substrates unambiguously confirm that it constitutes an important intermediary in the reaction.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cellular and Molecular Microbiology and Pathogenicity (MCMP), UMR 5097-CNRS, University Victor Segalen Bordeaux 2, Bordeaux, France.

ABSTRACT
IntI1 integrase is a tyrosine recombinase involved in the mobility of antibiotic resistance gene cassettes within bacterial class 1 integrons. Recent data have shown that its recombination specifically involves the bottom strand of the attC site, but the exact mechanism of the reaction is still unclear. An efficient in vitro assay is still required to better characterize the biochemical properties of the enzyme. In this report we describe for the first time an in vitro system partially reproducing the activity of a recombinant pure IntI1. This new assay, which constitutes the only available in vitro model of recombination by IntI1, was used to determine whether this enzyme might be the sole bacterial protein required for the recombination process. Results show that IntI1 possesses all the features needed for performing recombination between attI and attC sites. However, differences in the in vitro intermolecular recombination efficiencies were found according to the target sites and were correlated with DNA affinities of the enzyme but not with in vivo data. The differential affinity of the enzyme for each site, its capacity to bind to a single-stranded structure at the attC site and the recombination observed with single-stranded substrates unambiguously confirm that it constitutes an important intermediary in the reaction. Our data strongly suggest that the enzyme possesses all the functions for generating and/or recognizing this structure even in the absence of other cellular factors. Furthermore, the in vitro assay reported here constitutes a powerful tool for the analysis of the recombination steps catalyzed by IntI1, its structure-function studies and the search for specific inhibitors.

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In vitro DNA binding of IntI1 with free double-stranded attI1 (A) and attC (B) recombination sites.Free 5′ 32P radiolabeled dsDNA fragments containing recombination sites (0.1 pmoles) were incubated with purified IntI1 (1–10 pmoles) at 4°C for 20 min before electrophoresis on 1% agarose gel run at 50 V, for 2 hours at 4°C. Arrows indicate the protein-DNA complexes and F corresponds to free recombination sites.
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pone-0001315-g003: In vitro DNA binding of IntI1 with free double-stranded attI1 (A) and attC (B) recombination sites.Free 5′ 32P radiolabeled dsDNA fragments containing recombination sites (0.1 pmoles) were incubated with purified IntI1 (1–10 pmoles) at 4°C for 20 min before electrophoresis on 1% agarose gel run at 50 V, for 2 hours at 4°C. Arrows indicate the protein-DNA complexes and F corresponds to free recombination sites.

Mentions: To investigate the ability of IntI1 to interact with the target sites attI1 and attC, standard gel mobility shift assays were performed using two radiolabeled fragments containing either the double-stranded attI1 or the attC site (respectively attI1ds and attCds). As shown in figure 3A, the mobility of the DNA fragment carrying the attI1ds site was lowered in the presence of IntI1.


A new in vitro strand transfer assay for monitoring bacterial class 1 integron recombinase IntI1 activity.

Dubois V, Debreyer C, Litvak S, Quentin C, Parissi V - PLoS ONE (2007)

In vitro DNA binding of IntI1 with free double-stranded attI1 (A) and attC (B) recombination sites.Free 5′ 32P radiolabeled dsDNA fragments containing recombination sites (0.1 pmoles) were incubated with purified IntI1 (1–10 pmoles) at 4°C for 20 min before electrophoresis on 1% agarose gel run at 50 V, for 2 hours at 4°C. Arrows indicate the protein-DNA complexes and F corresponds to free recombination sites.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2117344&req=5

pone-0001315-g003: In vitro DNA binding of IntI1 with free double-stranded attI1 (A) and attC (B) recombination sites.Free 5′ 32P radiolabeled dsDNA fragments containing recombination sites (0.1 pmoles) were incubated with purified IntI1 (1–10 pmoles) at 4°C for 20 min before electrophoresis on 1% agarose gel run at 50 V, for 2 hours at 4°C. Arrows indicate the protein-DNA complexes and F corresponds to free recombination sites.
Mentions: To investigate the ability of IntI1 to interact with the target sites attI1 and attC, standard gel mobility shift assays were performed using two radiolabeled fragments containing either the double-stranded attI1 or the attC site (respectively attI1ds and attCds). As shown in figure 3A, the mobility of the DNA fragment carrying the attI1ds site was lowered in the presence of IntI1.

Bottom Line: Results show that IntI1 possesses all the features needed for performing recombination between attI and attC sites.However, differences in the in vitro intermolecular recombination efficiencies were found according to the target sites and were correlated with DNA affinities of the enzyme but not with in vivo data.The differential affinity of the enzyme for each site, its capacity to bind to a single-stranded structure at the attC site and the recombination observed with single-stranded substrates unambiguously confirm that it constitutes an important intermediary in the reaction.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cellular and Molecular Microbiology and Pathogenicity (MCMP), UMR 5097-CNRS, University Victor Segalen Bordeaux 2, Bordeaux, France.

ABSTRACT
IntI1 integrase is a tyrosine recombinase involved in the mobility of antibiotic resistance gene cassettes within bacterial class 1 integrons. Recent data have shown that its recombination specifically involves the bottom strand of the attC site, but the exact mechanism of the reaction is still unclear. An efficient in vitro assay is still required to better characterize the biochemical properties of the enzyme. In this report we describe for the first time an in vitro system partially reproducing the activity of a recombinant pure IntI1. This new assay, which constitutes the only available in vitro model of recombination by IntI1, was used to determine whether this enzyme might be the sole bacterial protein required for the recombination process. Results show that IntI1 possesses all the features needed for performing recombination between attI and attC sites. However, differences in the in vitro intermolecular recombination efficiencies were found according to the target sites and were correlated with DNA affinities of the enzyme but not with in vivo data. The differential affinity of the enzyme for each site, its capacity to bind to a single-stranded structure at the attC site and the recombination observed with single-stranded substrates unambiguously confirm that it constitutes an important intermediary in the reaction. Our data strongly suggest that the enzyme possesses all the functions for generating and/or recognizing this structure even in the absence of other cellular factors. Furthermore, the in vitro assay reported here constitutes a powerful tool for the analysis of the recombination steps catalyzed by IntI1, its structure-function studies and the search for specific inhibitors.

Show MeSH
Related in: MedlinePlus