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In vitro recombination catalyzed by bacterial class 1 integron integrase IntI1 involves cooperative binding and specific oligomeric intermediates.

Dubois V, Debreyer C, Quentin C, Parissi V - PLoS ONE (2009)

Bottom Line: This suggests a possible role for this protein in stabilizing and/or generating this structure.The mechanism of folding of the active IntI*DNA complexes was further analyzed and we show here that it involves a cooperative binding of the protein to each recombination site and the emergence of different oligomeric species specific for each DNA substrate.These findings provide further insight into the recombination reaction catalyzed by IntI1.

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
Gene transfer via bacterial integrons is a major pathway for facilitating the spread of antibiotic resistance genes across bacteria. Recently the mechanism underlying the recombination catalyzed by class 1 integron recombinase (IntI1) between attC and attI1 was highlighted demonstrating the involvement of a single-stranded intermediary on the attC site. However, the process allowing the generation of this single-stranded substrate has not been determined, nor have the active IntI1*DNA complexes been identified. Using the in vitro strand transfer assay and a crosslink strategy we previously described we demonstrated that the single-stranded attC sequences could be generated in the absence of other bacterial proteins in addition to IntI. This suggests a possible role for this protein in stabilizing and/or generating this structure. The mechanism of folding of the active IntI*DNA complexes was further analyzed and we show here that it involves a cooperative binding of the protein to each recombination site and the emergence of different oligomeric species specific for each DNA substrate. These findings provide further insight into the recombination reaction catalyzed by IntI1.

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In vitro DNA binding of IntI1 with double- or single-stranded attI1 (A) and attC (B) sites.Free 5′ 32P radiolabeled dsDNA fragments containing recombination sites (0.1 pmoles) were incubated with purified IntI1 (1–15 pmoles) at 4°C for 20 min before electrophoresis on 1% agarose gel run at 50 V, for 2 hours at 4°C. Gel shifted bands were then quantified using DNAJ software and are plotted in the figure as percentage of bound DNA. Results are the mean±standard deviation (error bars) of three independent experiments.
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pone-0005228-g002: In vitro DNA binding of IntI1 with double- or single-stranded attI1 (A) and attC (B) sites.Free 5′ 32P radiolabeled dsDNA fragments containing recombination sites (0.1 pmoles) were incubated with purified IntI1 (1–15 pmoles) at 4°C for 20 min before electrophoresis on 1% agarose gel run at 50 V, for 2 hours at 4°C. Gel shifted bands were then quantified using DNAJ software and are plotted in the figure as percentage of bound DNA. Results are the mean±standard deviation (error bars) of three independent experiments.

Mentions: Recombination activity catalyzed by IntI1 between the attI1 and attC sites requires correct binding of the protein on each substrate. However, differences in affinity were previously reported depending on the recombination sequence bound [11], [13], [14]. Gel shift assays performed with our recombinant protein showed that this enzyme shares the same DNA affinity reported previously. Indeed as shown in fig 2, IntI1 had a better affinity for the dsattI1 and the single-stranded bsattC while this enzyme only poorly bound the dsattC site.


In vitro recombination catalyzed by bacterial class 1 integron integrase IntI1 involves cooperative binding and specific oligomeric intermediates.

Dubois V, Debreyer C, Quentin C, Parissi V - PLoS ONE (2009)

In vitro DNA binding of IntI1 with double- or single-stranded attI1 (A) and attC (B) sites.Free 5′ 32P radiolabeled dsDNA fragments containing recombination sites (0.1 pmoles) were incubated with purified IntI1 (1–15 pmoles) at 4°C for 20 min before electrophoresis on 1% agarose gel run at 50 V, for 2 hours at 4°C. Gel shifted bands were then quantified using DNAJ software and are plotted in the figure as percentage of bound DNA. Results are the mean±standard deviation (error bars) of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2668188&req=5

pone-0005228-g002: In vitro DNA binding of IntI1 with double- or single-stranded attI1 (A) and attC (B) sites.Free 5′ 32P radiolabeled dsDNA fragments containing recombination sites (0.1 pmoles) were incubated with purified IntI1 (1–15 pmoles) at 4°C for 20 min before electrophoresis on 1% agarose gel run at 50 V, for 2 hours at 4°C. Gel shifted bands were then quantified using DNAJ software and are plotted in the figure as percentage of bound DNA. Results are the mean±standard deviation (error bars) of three independent experiments.
Mentions: Recombination activity catalyzed by IntI1 between the attI1 and attC sites requires correct binding of the protein on each substrate. However, differences in affinity were previously reported depending on the recombination sequence bound [11], [13], [14]. Gel shift assays performed with our recombinant protein showed that this enzyme shares the same DNA affinity reported previously. Indeed as shown in fig 2, IntI1 had a better affinity for the dsattI1 and the single-stranded bsattC while this enzyme only poorly bound the dsattC site.

Bottom Line: This suggests a possible role for this protein in stabilizing and/or generating this structure.The mechanism of folding of the active IntI*DNA complexes was further analyzed and we show here that it involves a cooperative binding of the protein to each recombination site and the emergence of different oligomeric species specific for each DNA substrate.These findings provide further insight into the recombination reaction catalyzed by IntI1.

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
Gene transfer via bacterial integrons is a major pathway for facilitating the spread of antibiotic resistance genes across bacteria. Recently the mechanism underlying the recombination catalyzed by class 1 integron recombinase (IntI1) between attC and attI1 was highlighted demonstrating the involvement of a single-stranded intermediary on the attC site. However, the process allowing the generation of this single-stranded substrate has not been determined, nor have the active IntI1*DNA complexes been identified. Using the in vitro strand transfer assay and a crosslink strategy we previously described we demonstrated that the single-stranded attC sequences could be generated in the absence of other bacterial proteins in addition to IntI. This suggests a possible role for this protein in stabilizing and/or generating this structure. The mechanism of folding of the active IntI*DNA complexes was further analyzed and we show here that it involves a cooperative binding of the protein to each recombination site and the emergence of different oligomeric species specific for each DNA substrate. These findings provide further insight into the recombination reaction catalyzed by IntI1.

Show MeSH
Related in: MedlinePlus