Limits...
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: 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.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

In vitro recombination catalyzed by wild type, R146E and R286K mutated IntI1.Reactions were performed for 90 min in the presence of purified enzyme (5 pmoles), 0.1 pmoles of linear radiolabeled recombination sites attI1 or attC and 0.1 pmoles of pGEM-T-attI1 under standard conditions described in materials and methods section. Products were loaded on 1% agarose gel and autoradiographied. F: free recombination sites, RP: recombination products.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2117344&req=5

pone-0001315-g007: In vitro recombination catalyzed by wild type, R146E and R286K mutated IntI1.Reactions were performed for 90 min in the presence of purified enzyme (5 pmoles), 0.1 pmoles of linear radiolabeled recombination sites attI1 or attC and 0.1 pmoles of pGEM-T-attI1 under standard conditions described in materials and methods section. Products were loaded on 1% agarose gel and autoradiographied. F: free recombination sites, RP: recombination products.

Mentions: To better ascertain the specificity of the in vitro recombination reaction catalyzed by IntI1, two mutated enzymes containing amino acid substitutions R146K and R280E were assayed. Those two invariant residues were demonstrated to be involved in the in vivo recombination activity. Since the two mutants were previously shown to be inactive for in vivo recombination activity [19], we analyzed their in vitro properties. DNA binding experiments (data not reported here) showed that only the R146K mutant presented an in vitro DNA binding property in the presence of attI1 but not in the presence of the attC element. The second R280E mutant showed no affinity at all for attI1 or attC as previously described [22]. The in vitro activities of both mutants were then assayed for both attI x attI and attI x attC recombinations. As shown in figure 7, no activity was detected in either mutant. These results demonstrate firstly that the two amino acids R146 and R280 are required for recombination catalysis, and that the reaction observed with wild type IntI1 was due to the intrinsic catalytic capability of the recombinant integrase.


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 recombination catalyzed by wild type, R146E and R286K mutated IntI1.Reactions were performed for 90 min in the presence of purified enzyme (5 pmoles), 0.1 pmoles of linear radiolabeled recombination sites attI1 or attC and 0.1 pmoles of pGEM-T-attI1 under standard conditions described in materials and methods section. Products were loaded on 1% agarose gel and autoradiographied. F: free recombination sites, RP: recombination products.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0001315-g007: In vitro recombination catalyzed by wild type, R146E and R286K mutated IntI1.Reactions were performed for 90 min in the presence of purified enzyme (5 pmoles), 0.1 pmoles of linear radiolabeled recombination sites attI1 or attC and 0.1 pmoles of pGEM-T-attI1 under standard conditions described in materials and methods section. Products were loaded on 1% agarose gel and autoradiographied. F: free recombination sites, RP: recombination products.
Mentions: To better ascertain the specificity of the in vitro recombination reaction catalyzed by IntI1, two mutated enzymes containing amino acid substitutions R146K and R280E were assayed. Those two invariant residues were demonstrated to be involved in the in vivo recombination activity. Since the two mutants were previously shown to be inactive for in vivo recombination activity [19], we analyzed their in vitro properties. DNA binding experiments (data not reported here) showed that only the R146K mutant presented an in vitro DNA binding property in the presence of attI1 but not in the presence of the attC element. The second R280E mutant showed no affinity at all for attI1 or attC as previously described [22]. The in vitro activities of both mutants were then assayed for both attI x attI and attI x attC recombinations. As shown in figure 7, no activity was detected in either mutant. These results demonstrate firstly that the two amino acids R146 and R280 are required for recombination catalysis, and that the reaction observed with wild type IntI1 was due to the intrinsic catalytic capability of the recombinant integrase.

Bottom Line: 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.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