Reactions of Cre with Methylphosphonate DNA: Similarities and Contrasts with Flp and Vaccinia Topoisomerase

Abstract

BackgroundReactions 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 FindingsWe 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/SignificanceCollectively, 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.

Electrostatic Suppression Allows Tyrosine Site-specific Recombination in the Absence of a Conserved Catalytic Arginine

“…Type I activity is evident in a Flp/P half-site reaction and is detectable but weak in a Flp/ MeP half-site reaction (this study and Ref. 13). Experiments in the present work have examined the effects of replacing the second arginine of the pentad, Arg-191, by alanine and the responses of the variant Flp to MeP-DNA.…”

“…This lack of reaction is consistent with the very poor formation of the tyrosyl intermediate by Flp and Flp(R191A) on the MeP and P substrates, respectively (Ref. 13 and this study). When the Flp(R191A)-bound half-site was reacted with Flp and the P full-site, the predicted exchange products from the alternative site alignments were formed (Fig.…”

“…Image analysis was performed using the software Quantity One, version 4.5.1 (Bio-Rad). Nonlinear Regression Curve Fitting-Data analysis was performed using the software GraphPad Prism (version 5.02) as previously described (13).…”

“…Recent studies show that substitution of the scissile phosphate (P) 3 by methylphosphonate (MeP), which is charge neutral in the ground state and carries a nominal Ϫ1 charge in the transition state, makes Arg-308 dispensable in the MeP activation step by Flp (11). The same holds for the corresponding arginine residues of the Cre recombinase (Arg-292) and vaccinia topoisomerase (Arg-223) (12,13). The chemical competence of Cre(R292A), Flp(R308A), and Topo(R223A) on their respective MeP substrates is consistent with the suspected electrophilic role of the positively charged arginine side chain in catalysis.…”

“…Such a dramatic difference is not observed between the hydrolysis rates of P-tyrosyl and MeP-tyrosyl intermediates by Cre (2.8 ϫ 10 Ϫ5 s Ϫ1 ) and Cre(R292A) (8.8 ϫ 10 Ϫ5 s Ϫ1 ; racemic mixture of S P /R P MeP), respectively (13). The endonucleolytic activity of Cre and topoisomerase, directed to the covalent intermediate resulting from strand cleavage, is referred to as type I endonuclease activity (11,13).…”