Limits...
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.

Show MeSH

Related in: MedlinePlus

Model for the cooperative binding and the inti1 oligomers involved in the in vitro recombination catalyzed by IntI1.In the attI1×attI1 recombination reaction IntI1 binds both dsattI1 fragment as a dimer and catalyzes the strand exchange and the formation of the HJ intermediate (lanes A1 to A3). In the case of attI1×attC recombination, IntI1 binds dsattC only slightly (B1). Interaction with the first attI1 substrate led to cooperative binding to the second attC site (B2), allowing the recruitment of a second IntI1 dimer on the second strand of the attC site (B3) and the formation of the tetrameric intermediate, leading in turn to the stabilization of a ssattC intermediate (B4). The strand exchange between dsattI1 and bsattC can then be catalyzed (B5). Recombination activity detected in presence of two attC sites suggests that the initial low binding of the enzyme to this site is sufficient for triggering all the subsequent recombination steps (way C). ts: top strand, bs: bottom strand.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2668188&req=5

pone-0005228-g005: Model for the cooperative binding and the inti1 oligomers involved in the in vitro recombination catalyzed by IntI1.In the attI1×attI1 recombination reaction IntI1 binds both dsattI1 fragment as a dimer and catalyzes the strand exchange and the formation of the HJ intermediate (lanes A1 to A3). In the case of attI1×attC recombination, IntI1 binds dsattC only slightly (B1). Interaction with the first attI1 substrate led to cooperative binding to the second attC site (B2), allowing the recruitment of a second IntI1 dimer on the second strand of the attC site (B3) and the formation of the tetrameric intermediate, leading in turn to the stabilization of a ssattC intermediate (B4). The strand exchange between dsattI1 and bsattC can then be catalyzed (B5). Recombination activity detected in presence of two attC sites suggests that the initial low binding of the enzyme to this site is sufficient for triggering all the subsequent recombination steps (way C). ts: top strand, bs: bottom strand.

Mentions: Taken together, our data in addition to previous reports allow us to propose a molecular model for the in vitro recombination catalyzed by IntI1 (fig 5). In the attI1×attI1 recombination reaction, IntI1 binds both dsattI1 fragments under dimeric form and catalyzes the usual strand exchange and the formation of the HJ intermediate (way A1 to A3). In the case of attI1×attC recombination, IntI1 binds dsattC only slightly (B1). Interaction with the first attI1 substrate leads to cooperative binding to the second attC site (B2), thus allowing the recruitment of a second IntI1 dimer on the second strand of the attC site (B3) and the formation of the tetrameric intermediate, which in turn stabilizes a ssattC structure (B4) leading to the catalysis of the strand exchange between dsattI1 and bsattC (B5). Recombination activity detected in the presence of two attC sites suggests that the initial low binding of the enzyme to the site is sufficient to trigger all the subsequent recombination steps (way C). It is important to point out that the work reported here leading to this model accounts only for the recombination activity the cooperative binding and the oligomerization state of the protein on each substrate detected in vitro. The precise structure of each intermediate remains to be elucidated, especially the tetrameric complex formed with the dsattC site. In addition, the process underlying the generation of the ssattC fragment also remains to be established and is under study in the laboratory.


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)

Model for the cooperative binding and the inti1 oligomers involved in the in vitro recombination catalyzed by IntI1.In the attI1×attI1 recombination reaction IntI1 binds both dsattI1 fragment as a dimer and catalyzes the strand exchange and the formation of the HJ intermediate (lanes A1 to A3). In the case of attI1×attC recombination, IntI1 binds dsattC only slightly (B1). Interaction with the first attI1 substrate led to cooperative binding to the second attC site (B2), allowing the recruitment of a second IntI1 dimer on the second strand of the attC site (B3) and the formation of the tetrameric intermediate, leading in turn to the stabilization of a ssattC intermediate (B4). The strand exchange between dsattI1 and bsattC can then be catalyzed (B5). Recombination activity detected in presence of two attC sites suggests that the initial low binding of the enzyme to this site is sufficient for triggering all the subsequent recombination steps (way C). ts: top strand, bs: bottom strand.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0005228-g005: Model for the cooperative binding and the inti1 oligomers involved in the in vitro recombination catalyzed by IntI1.In the attI1×attI1 recombination reaction IntI1 binds both dsattI1 fragment as a dimer and catalyzes the strand exchange and the formation of the HJ intermediate (lanes A1 to A3). In the case of attI1×attC recombination, IntI1 binds dsattC only slightly (B1). Interaction with the first attI1 substrate led to cooperative binding to the second attC site (B2), allowing the recruitment of a second IntI1 dimer on the second strand of the attC site (B3) and the formation of the tetrameric intermediate, leading in turn to the stabilization of a ssattC intermediate (B4). The strand exchange between dsattI1 and bsattC can then be catalyzed (B5). Recombination activity detected in presence of two attC sites suggests that the initial low binding of the enzyme to this site is sufficient for triggering all the subsequent recombination steps (way C). ts: top strand, bs: bottom strand.
Mentions: Taken together, our data in addition to previous reports allow us to propose a molecular model for the in vitro recombination catalyzed by IntI1 (fig 5). In the attI1×attI1 recombination reaction, IntI1 binds both dsattI1 fragments under dimeric form and catalyzes the usual strand exchange and the formation of the HJ intermediate (way A1 to A3). In the case of attI1×attC recombination, IntI1 binds dsattC only slightly (B1). Interaction with the first attI1 substrate leads to cooperative binding to the second attC site (B2), thus allowing the recruitment of a second IntI1 dimer on the second strand of the attC site (B3) and the formation of the tetrameric intermediate, which in turn stabilizes a ssattC structure (B4) leading to the catalysis of the strand exchange between dsattI1 and bsattC (B5). Recombination activity detected in the presence of two attC sites suggests that the initial low binding of the enzyme to the site is sufficient to trigger all the subsequent recombination steps (way C). It is important to point out that the work reported here leading to this model accounts only for the recombination activity the cooperative binding and the oligomerization state of the protein on each substrate detected in vitro. The precise structure of each intermediate remains to be elucidated, especially the tetrameric complex formed with the dsattC site. In addition, the process underlying the generation of the ssattC fragment also remains to be established and is under study in the laboratory.

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