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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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Lack of endonucleolytic activity in MeP-half-site reactions of Cre(R292A, Y324G).Reactions and product analysis were performed as described under Figure 3. The results of SDS-PAGE and denaturing PAGE analyses are shown in A and B, respectively. The predicted positions of migration of the covalent intermediate (line ending in a circular knob) and the hydrolysis product (HP) are indicated. There was a faint band of background hydrolysis in the control reaction, which did not increase in intensity with increasing time of incubation with Cre.
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pone-0007248-g005: Lack of endonucleolytic activity in MeP-half-site reactions of Cre(R292A, Y324G).Reactions and product analysis were performed as described under Figure 3. The results of SDS-PAGE and denaturing PAGE analyses are shown in A and B, respectively. The predicted positions of migration of the covalent intermediate (line ending in a circular knob) and the hydrolysis product (HP) are indicated. There was a faint band of background hydrolysis in the control reaction, which did not increase in intensity with increasing time of incubation with Cre.

Mentions: The kinetic data in Figure 4 are consistent with the 24-mer being the hydrolytic breakdown product of the MeP-tyrosyl intermediate. In accordance with their precursor-product relationship, treatment of the MeP-half-site with Cre(R292A,Y324G) did not result in the formation of either the cleaved intermediate or the 24-mer (Figure 5).


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)

Lack of endonucleolytic activity in MeP-half-site reactions of Cre(R292A, Y324G).Reactions and product analysis were performed as described under Figure 3. The results of SDS-PAGE and denaturing PAGE analyses are shown in A and B, respectively. The predicted positions of migration of the covalent intermediate (line ending in a circular knob) and the hydrolysis product (HP) are indicated. There was a faint band of background hydrolysis in the control reaction, which did not increase in intensity with increasing time of incubation with Cre.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0007248-g005: Lack of endonucleolytic activity in MeP-half-site reactions of Cre(R292A, Y324G).Reactions and product analysis were performed as described under Figure 3. The results of SDS-PAGE and denaturing PAGE analyses are shown in A and B, respectively. The predicted positions of migration of the covalent intermediate (line ending in a circular knob) and the hydrolysis product (HP) are indicated. There was a faint band of background hydrolysis in the control reaction, which did not increase in intensity with increasing time of incubation with Cre.
Mentions: The kinetic data in Figure 4 are consistent with the 24-mer being the hydrolytic breakdown product of the MeP-tyrosyl intermediate. In accordance with their precursor-product relationship, treatment of the MeP-half-site with Cre(R292A,Y324G) did not result in the formation of either the cleaved intermediate or the 24-mer (Figure 5).

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