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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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Mechanisms for the protection of the transesterification steps of DNA relaxation and site-specific recombination from hydrolysis.A, B. The strand cleavage steps of DNA relaxation by topoisomerase and recombination by Cre are protected by the temporal coupling between protein-DNA association and engagement of the scissile phosphate by the cis-acting active site. C. Binding of a Flp monomer to DNA activates the scissile phosphate but full engagement of the active site must await the binding of a second Flp monomer and donation of the active site tyrosine. Hydrolysis of the phosphodiester bond becomes a potential risk, which is minimized through active site electrostatics. D. During topoisomerase action, the cleaved tyrosyl intermediate is a potential target for attack by water during the strand rotation step that precedes ligation. The danger of hydrolysis at this stage is avoided through phosphate electrostatics.
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pone-0007248-g007: Mechanisms for the protection of the transesterification steps of DNA relaxation and site-specific recombination from hydrolysis.A, B. The strand cleavage steps of DNA relaxation by topoisomerase and recombination by Cre are protected by the temporal coupling between protein-DNA association and engagement of the scissile phosphate by the cis-acting active site. C. Binding of a Flp monomer to DNA activates the scissile phosphate but full engagement of the active site must await the binding of a second Flp monomer and donation of the active site tyrosine. Hydrolysis of the phosphodiester bond becomes a potential risk, which is minimized through active site electrostatics. D. During topoisomerase action, the cleaved tyrosyl intermediate is a potential target for attack by water during the strand rotation step that precedes ligation. The danger of hydrolysis at this stage is avoided through phosphate electrostatics.

Mentions: Topoisomerase binds to DNA and relaxes it as a monomer. Flp and Cre also bind each of the two binding elements on their target sites as monomers. Because of their cis configuration, the topoisomerase and Cre active sites engage the scissile phosphodiesters contemporaneous to DNA binding. This would leave little chance for water to channel the cleavage reaction towards hydrolysis (Figure 7A and B). Whereas the topoisomerase monomer is fully competent for strand cleavage, the Cre monomer likely requires allosteric activation by a neighboring monomer to become cleavage proficient [3], [6].


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)

Mechanisms for the protection of the transesterification steps of DNA relaxation and site-specific recombination from hydrolysis.A, B. The strand cleavage steps of DNA relaxation by topoisomerase and recombination by Cre are protected by the temporal coupling between protein-DNA association and engagement of the scissile phosphate by the cis-acting active site. C. Binding of a Flp monomer to DNA activates the scissile phosphate but full engagement of the active site must await the binding of a second Flp monomer and donation of the active site tyrosine. Hydrolysis of the phosphodiester bond becomes a potential risk, which is minimized through active site electrostatics. D. During topoisomerase action, the cleaved tyrosyl intermediate is a potential target for attack by water during the strand rotation step that precedes ligation. The danger of hydrolysis at this stage is avoided through phosphate electrostatics.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0007248-g007: Mechanisms for the protection of the transesterification steps of DNA relaxation and site-specific recombination from hydrolysis.A, B. The strand cleavage steps of DNA relaxation by topoisomerase and recombination by Cre are protected by the temporal coupling between protein-DNA association and engagement of the scissile phosphate by the cis-acting active site. C. Binding of a Flp monomer to DNA activates the scissile phosphate but full engagement of the active site must await the binding of a second Flp monomer and donation of the active site tyrosine. Hydrolysis of the phosphodiester bond becomes a potential risk, which is minimized through active site electrostatics. D. During topoisomerase action, the cleaved tyrosyl intermediate is a potential target for attack by water during the strand rotation step that precedes ligation. The danger of hydrolysis at this stage is avoided through phosphate electrostatics.
Mentions: Topoisomerase binds to DNA and relaxes it as a monomer. Flp and Cre also bind each of the two binding elements on their target sites as monomers. Because of their cis configuration, the topoisomerase and Cre active sites engage the scissile phosphodiesters contemporaneous to DNA binding. This would leave little chance for water to channel the cleavage reaction towards hydrolysis (Figure 7A and B). Whereas the topoisomerase monomer is fully competent for strand cleavage, the Cre monomer likely requires allosteric activation by a neighboring monomer to become cleavage proficient [3], [6].

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