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Reactions of Cre with methylphosphonate DNA: similarities and contrasts with Flp and vaccinia topoisomerase.

Ma CH, Kachroo AH, Macieszak A, Chen TY, Guga P, Jayaram M - PLoS ONE (2009)

Bottom Line: Neutralizing the negative charge at the scissile position does not render the tyrosyl intermediate formed by Cre susceptible to rapid hydrolysis.Furthermore, combining the active site R292A mutation in Cre (equivalent to the R223A and R308A mutations in topoisomerase and Flp, respectively) with MeP substitution does not lead to direct hydrolysis of the scissile MeP bond in DNA.Such protective mechanisms are significant, given the very real threat of hydrolytic genome damage or disruption of RNA processing due to the cellular abundance and nucleophilicity of water.

View Article: PubMed Central - PubMed

Affiliation: Section of Molecular Genetics and Microbiology, University of Texas at Austin, Austin, Texas, United States of America.

ABSTRACT

Background: Reactions of vaccinia topoisomerase and the tyrosine site-specific recombinase Flp with methylphosphonate (MeP) substituted DNA substrates, have provided important insights into the electrostatic features of the strand cleavage and strand joining steps catalyzed by them. A conserved arginine residue in the catalytic pentad, Arg-223 in topoisomerase and Arg-308 in Flp, is not essential for stabilizing the MeP transition state. Topoisomerase or its R223A variant promotes cleavage of the MeP bond by the active site nucleophile Tyr-274, followed by the rapid hydrolysis of the MeP-tyrosyl intermediate. Flp(R308A), but not wild type Flp, mediates direct hydrolysis of the activated MeP bond. These findings are consistent with a potential role for phosphate electrostatics and active site electrostatics in protecting DNA relaxation and site-specific recombination, respectively, against abortive hydrolysis.

Methodology/principal findings: We have examined the effects of DNA containing MeP substitution in the Flp related Cre recombination system. Neutralizing the negative charge at the scissile position does not render the tyrosyl intermediate formed by Cre susceptible to rapid hydrolysis. Furthermore, combining the active site R292A mutation in Cre (equivalent to the R223A and R308A mutations in topoisomerase and Flp, respectively) with MeP substitution does not lead to direct hydrolysis of the scissile MeP bond in DNA. Whereas Cre follows the topoisomerase paradigm during the strand cleavage step, it follows the Flp paradigm during the strand joining step.

Conclusions/significance: Collectively, the Cre, Flp and topoisomerase results highlight the contribution of conserved electrostatic complementarity between substrate and active site towards transition state stabilization during site-specific recombination and DNA relaxation. They have potential implications for how transesterification reactions in nucleic acids are protected against undesirable abortive side reactions. Such protective mechanisms are significant, given the very real threat of hydrolytic genome damage or disruption of RNA processing due to the cellular abundance and nucleophilicity of water.

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Strand cleavage and endonuclease activities of Cre in P- and MeP-half-site substrates.In the schematic representations of half-site substrates, the asterisk indicates 32P-label at the 5′-end, ‘p’ the scissile phosphate and ‘mp’ the scissile methylphosphonate. Reactions were split into halves to analyze the covalent Cre-DNA adduct (line ending in a circular knob) by SDS-PAGE (A) and the potential hydrolysis product(s) by denaturing PAGE (B). Reactions of Flp with its cognate half-sites in similar assays [19] are provided for comparison. The end-labeled half-site (A) or its labeled strand (B) is denoted by ‘S’ and the hydrolysis product by ‘HP’. The labeled strands in the Cre and Flp half-sites were 27 and 26 nucleotides long, respectively. HP in the Cre reaction was a 24-mer and that in the Flp reaction a 23-mer. The plots below represent mean values from three separate experiments with Cre.
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pone-0007248-g003: Strand cleavage and endonuclease activities of Cre in P- and MeP-half-site substrates.In the schematic representations of half-site substrates, the asterisk indicates 32P-label at the 5′-end, ‘p’ the scissile phosphate and ‘mp’ the scissile methylphosphonate. Reactions were split into halves to analyze the covalent Cre-DNA adduct (line ending in a circular knob) by SDS-PAGE (A) and the potential hydrolysis product(s) by denaturing PAGE (B). Reactions of Flp with its cognate half-sites in similar assays [19] are provided for comparison. The end-labeled half-site (A) or its labeled strand (B) is denoted by ‘S’ and the hydrolysis product by ‘HP’. The labeled strands in the Cre and Flp half-sites were 27 and 26 nucleotides long, respectively. HP in the Cre reaction was a 24-mer and that in the Flp reaction a 23-mer. The plots below represent mean values from three separate experiments with Cre.

Mentions: The rate constants for strand cleavage (kcl) and hydrolysis of the tyrosyl intermediate (khydrol) were estimated from data shown in Figures 3 and 4 and additional data from similar assays. The software package Prism (version 5.02) for Windows (GraphPad Software, Inc.) was used for obtaining the kinetic parameters. In the reaction scheme the first step of strand cleavage and formation of the Cre-DNA adduct is assumed to be irreversible. The trinucleotide product resulting from cleavage would diffuse away, and be unavailable for the back reaction. The values for the rate constants for Flp and vaccinia topoisomerase are taken from published work [19], [20].


Reactions of Cre with methylphosphonate DNA: similarities and contrasts with Flp and vaccinia topoisomerase.

Ma CH, Kachroo AH, Macieszak A, Chen TY, Guga P, Jayaram M - PLoS ONE (2009)

Strand cleavage and endonuclease activities of Cre in P- and MeP-half-site substrates.In the schematic representations of half-site substrates, the asterisk indicates 32P-label at the 5′-end, ‘p’ the scissile phosphate and ‘mp’ the scissile methylphosphonate. Reactions were split into halves to analyze the covalent Cre-DNA adduct (line ending in a circular knob) by SDS-PAGE (A) and the potential hydrolysis product(s) by denaturing PAGE (B). Reactions of Flp with its cognate half-sites in similar assays [19] are provided for comparison. The end-labeled half-site (A) or its labeled strand (B) is denoted by ‘S’ and the hydrolysis product by ‘HP’. The labeled strands in the Cre and Flp half-sites were 27 and 26 nucleotides long, respectively. HP in the Cre reaction was a 24-mer and that in the Flp reaction a 23-mer. The plots below represent mean values from three separate experiments with Cre.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2747268&req=5

pone-0007248-g003: Strand cleavage and endonuclease activities of Cre in P- and MeP-half-site substrates.In the schematic representations of half-site substrates, the asterisk indicates 32P-label at the 5′-end, ‘p’ the scissile phosphate and ‘mp’ the scissile methylphosphonate. Reactions were split into halves to analyze the covalent Cre-DNA adduct (line ending in a circular knob) by SDS-PAGE (A) and the potential hydrolysis product(s) by denaturing PAGE (B). Reactions of Flp with its cognate half-sites in similar assays [19] are provided for comparison. The end-labeled half-site (A) or its labeled strand (B) is denoted by ‘S’ and the hydrolysis product by ‘HP’. The labeled strands in the Cre and Flp half-sites were 27 and 26 nucleotides long, respectively. HP in the Cre reaction was a 24-mer and that in the Flp reaction a 23-mer. The plots below represent mean values from three separate experiments with Cre.
Mentions: The rate constants for strand cleavage (kcl) and hydrolysis of the tyrosyl intermediate (khydrol) were estimated from data shown in Figures 3 and 4 and additional data from similar assays. The software package Prism (version 5.02) for Windows (GraphPad Software, Inc.) was used for obtaining the kinetic parameters. In the reaction scheme the first step of strand cleavage and formation of the Cre-DNA adduct is assumed to be irreversible. The trinucleotide product resulting from cleavage would diffuse away, and be unavailable for the back reaction. The values for the rate constants for Flp and vaccinia topoisomerase are taken from published work [19], [20].

Bottom Line: Neutralizing the negative charge at the scissile position does not render the tyrosyl intermediate formed by Cre susceptible to rapid hydrolysis.Furthermore, combining the active site R292A mutation in Cre (equivalent to the R223A and R308A mutations in topoisomerase and Flp, respectively) with MeP substitution does not lead to direct hydrolysis of the scissile MeP bond in DNA.Such protective mechanisms are significant, given the very real threat of hydrolytic genome damage or disruption of RNA processing due to the cellular abundance and nucleophilicity of water.

View Article: PubMed Central - PubMed

Affiliation: Section of Molecular Genetics and Microbiology, University of Texas at Austin, Austin, Texas, United States of America.

ABSTRACT

Background: Reactions of vaccinia topoisomerase and the tyrosine site-specific recombinase Flp with methylphosphonate (MeP) substituted DNA substrates, have provided important insights into the electrostatic features of the strand cleavage and strand joining steps catalyzed by them. A conserved arginine residue in the catalytic pentad, Arg-223 in topoisomerase and Arg-308 in Flp, is not essential for stabilizing the MeP transition state. Topoisomerase or its R223A variant promotes cleavage of the MeP bond by the active site nucleophile Tyr-274, followed by the rapid hydrolysis of the MeP-tyrosyl intermediate. Flp(R308A), but not wild type Flp, mediates direct hydrolysis of the activated MeP bond. These findings are consistent with a potential role for phosphate electrostatics and active site electrostatics in protecting DNA relaxation and site-specific recombination, respectively, against abortive hydrolysis.

Methodology/principal findings: We have examined the effects of DNA containing MeP substitution in the Flp related Cre recombination system. Neutralizing the negative charge at the scissile position does not render the tyrosyl intermediate formed by Cre susceptible to rapid hydrolysis. Furthermore, combining the active site R292A mutation in Cre (equivalent to the R223A and R308A mutations in topoisomerase and Flp, respectively) with MeP substitution does not lead to direct hydrolysis of the scissile MeP bond in DNA. Whereas Cre follows the topoisomerase paradigm during the strand cleavage step, it follows the Flp paradigm during the strand joining step.

Conclusions/significance: Collectively, the Cre, Flp and topoisomerase results highlight the contribution of conserved electrostatic complementarity between substrate and active site towards transition state stabilization during site-specific recombination and DNA relaxation. They have potential implications for how transesterification reactions in nucleic acids are protected against undesirable abortive side reactions. Such protective mechanisms are significant, given the very real threat of hydrolytic genome damage or disruption of RNA processing due to the cellular abundance and nucleophilicity of water.

Show MeSH
Related in: MedlinePlus