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Chemical mutagenesis of vaccinia DNA topoisomerase lysine 167 provides insights to the catalysis of DNA transesterification.

ABSTRACT: Vaccinia DNA topoisomerase IB (TopIB) relaxes supercoils by forming and resealing a covalent DNA-(3'-phosphotyrosyl(274))-enzyme intermediate. Conserved active site side chains promote the attack of Tyr274 on the scissile phosphodiester via transition state stabilization and general acid catalysis. Two essential side chains, Lys167 and Arg130, act in concert to protonate and expel the 5'-O leaving group. Here we gained new insights to catalysis through chemical mutagenesis of Lys167. Changing Lys167 to cysteine crippled the DNA cleavage and religation transesterification steps (k(cl) = 4.3 × 10(-4) s(-1); k(rel) = 9 × 10(-4) s(-1)). The transesterification activities of the K167C enzyme were revived by in vitro alkylation with 2-bromoethylamine (k(cl) = 0.031 s(-1); k(rel) ? 0.4 s(-1)) and 3-bromopropylamine (k(cl) = 0.013 s(-1); k(rel) = 0.22 s(-1)), which convert the cysteines to ?-thialysine and ?-thiahomolysine, respectively. These chemically installed lysine analogues were more effective than a genetically programmed arginine 167 substitution characterized previously. The modest differences in the transesterification rates of the 2-bromoethylamine- and 3-bromopropylamine-treated enzymes highlight that TopIB is tolerant of a longer homolysine side chain for assembly of the active site and formation of the transition state.

SUBMITTER: Yakovleva L

PROVIDER: S-EPMC3619185 | biostudies-literature | 2013 Feb

REPOSITORIES: biostudies-literature

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Chemical mutagenesis of vaccinia DNA topoisomerase lysine 167 provides insights to the catalysis of DNA transesterification.

Yakovleva Lyudmila L Shuman Stewart S

Biochemistry 20130123 5

Vaccinia DNA topoisomerase IB (TopIB) relaxes supercoils by forming and resealing a covalent DNA-(3'-phosphotyrosyl(274))-enzyme intermediate. Conserved active site side chains promote the attack of Tyr274 on the scissile phosphodiester via transition state stabilization and general acid catalysis. Two essential side chains, Lys167 and Arg130, act in concert to protonate and expel the 5'-O leaving group. Here we gained new insights to catalysis through chemical mutagenesis of Lys167. Changing Ly ...[more]

PMID: 23317114

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Project description:The type I DNA topoisomerase from vaccinia virus (vTopo) forms a reversible covalent 3'-phosphotyrosyl linkage with a single strand of duplex DNA at the preferred sequence 5'-(C/T)CCTTp downward arrowN(-1)N(-2)N(-3)-3'. The enzyme-DNA covalent adduct is recombinogenic in cells, because the nicked strand downstream of the cleavage site can dissociate and be replaced by another DNA strand, potentially resulting in genome rearrangements if the enzyme executes strand ligation. Topo I could play an active role in strand exchange, either by altering the kinetics or thermodynamics of DNA strand binding or by serving as a proofreading gate to prevent ligation of incoming DNA strands containing mismatches. To address these questions, we have measured the kinetic and thermodynamic parameters for strand annealing to a purified vaccinia Topo I-DNA (vTopo-DNA) covalent complex containing a single-strand overhang and then compared them with the same overhang duplex in the absence of vTopo. We found that vTopo accelerates the strand association rate by 2-fold but has no effect on the rate of strand dissociation. vTopo has a similar small effect on the annealing parameters of a series of DNA strands containing single mismatches. In contrast, single base mismatches at the -1, -2, or -3 positions decreased the forward rate and equilibrium constant for reversible strand ligation by 10-fold. These data establish that while vTopo is a bystander during the annealing step of strand exchange, the enzyme strongly discriminates against mismatches close to the cleavage site during the subsequent events leading to strand ligation. A mechanism emerges where vTopo oscillates between an open state where the downstream DNA segment does not interact with the enzyme and a closed state where catalytically important contacts are formed with this region. This oscillation between an open and closed state of the covalently bound enzyme is likely important for regulating the number of DNA superhelical turns that are removed during the lifetime of the covalent complex with supercoiled substrates.

Project description: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.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.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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Chemical mutagenesis of vaccinia DNA topoisomerase lysine 167 provides insights to the catalysis of DNA transesterification.

Publications

Chemical mutagenesis of vaccinia DNA topoisomerase lysine 167 provides insights to the catalysis of DNA transesterification.

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