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Guarding the Genome
Molecular Cell, Vol. 12, 199–208, July, 2003, Copyright 2003 by Cell Press
Guarding the Genome: Electrostatic Repulsion of Water by DNA Suppresses a Potent Nuclease Activity of Topoisomerase IB Ligeng Tian,1 Christopher D. Claeboe,2 Sidney M. Hecht,2 and Stewart Shuman1,* 1 Molecular Biology Program Sloan-Kettering Institute New York, New York 10021 2 Departments of Chemistry and Biology University of Virginia Charlottesville, Virginia 22901
Summary Type IB topoisomerases cleave and rejoin DNA strands through a stable covalent DNA-(3ⴕ-phosphotyrosyl)-enzyme intermediate. The stability of the intermediate is a two-edged sword; it preserves genome integrity during supercoil relaxation, but it also reinforces the toxicity of drugs and lesions that interfere with the DNA rejoining step. Here, we identify a key determinant of the stability of the complex by showing that introduction of an Sp or Rp methylphosphonate linkage at the cleavage site transforms topoisomerase IB into a potent endonuclease. The nuclease reaction entails formation and surprisingly rapid hydrolysis of a covalent enzyme-DNA methylphosphonate intermediate. The ⵑ30,000-fold acceleration in the rate of hydrolysis of a methylphosphonate versus phosphodiester suggests that repulsion of water by the DNA phosphate anion suppresses the latent nuclease function of topoisomerase IB. These findings expose an Achilles’ heel of topoisomerases as guardians of the genome, and they have broad implications for understanding enzymatic phosphoryl transfer. Introduction Type IB topoisomerases alter DNA topology via a multistep reaction pathway entailing noncovalent binding of the enzyme to duplex DNA, cleavage of one DNA strand with formation of a covalent DNA-(3⬘-phosphotyrosyl)-protein intermediate, strand passage, and strand religation (Champoux, 2001; Shuman, 1998). The topoisomerase-DNA intermediate is extraordinarily stable at neutral pH. For example, vaccinia topoisomerase remains covalently bound to DNA for many days after “suicide cleavage” and remains fully competent to transfer the covalently held strand once it is provided with a 5⬘-OH acceptor DNA. The tyrosine-phosphodiester bond resists hydrolysis, even though the active site is accessible to solvent (Petersen and Shuman, 1997a; Krogh and Shuman, 2000b). The stability of the topoisomerase-DNA intermediate can be either a virtue or an Achilles’ heel, depending on the biological context. During normal topoisomerase function, which entails reiterative cleavage-ligation cycles at many sites in genomic DNA, it is important that the enzyme not relinquish its covalent hold on the 3⬘ *Correspondence: [email protected]
end of the nicked strand. To do so would abort the reaction and generate nicks that could be converted to lethal DNA breaks. In other situations, topoisomerase IB can become trapped in the covalent state, either by DNA lesions located at or 3⬘ to a cleavage site or by exposure to therapeutic drugs such as camptothecin (Burgin et al., 1995; Krogh et al., 1999; Lanza et al., 1996; Henningfield et al., 1996; Pourquier et al., 1997, 1998, 1999; Pommier et al., 2000, 2002; Hsiang et al., 1985; Staker et al., 2002). Such lesions and drugs selectively preclude the religation step by perturbing the position or reactivity of the DNA 5⬘-OH nucleophile with the DNA(3⬘-phosphotyrosyl)-topoisomerase complex. The covalent protein-DNA complex is itself a toxic lesion, and the toxicity is enforced by reluctance of the complex to undergo hydrolysis. In this setting, a nuclease activity associated with topoisomerase IB might be advantageous, allowing removal of topoisomerase from the DNA to generate a 3⬘-PO4/5-OH nick that can be repaired by the enzymes DNA 3⬘-phosphatase, DNA 5⬘-kinase, and DNA ligase. In vitro, topoisomerase IB can use water as a nucleophile to cleave the covalent intermediate and release a free 3⬘-PO4 DNA end, but the reaction is extremely slow and depends on nonphysiological alkaline pH conditions (Christiansen et al., 1994; Petersen and Shuman, 1997a). This reaction is not a reflection of the inherent chemical lability of the tyrosine phosphodiester; rather, hydrolysis is directly catalyzed by the topoisomerase active site (Krogh and Shuman, 2000b). Why then is topoisomerase not normally a more vigorous nuclease? An unexpected insight to this question emerges from an analysis of the effects of methylphosphonate modification of the scissile phosphodiester. We find that introduction of a methylphosphonate at the cleavage site transforms vaccinia topoisomerase into a potent site-specific endonuclease at physiological pH. The endonuclease reaction entails formation and rapid hydrolysis of a covalent enzyme-DNA methylphosphonate intermediate. Our results require a revised view of the catalytic mechanism of topoisomerase IB. The fact that transesterification occurs on the methylphosphonate DNAs attests to the lack of a stringent requirement for electrostatic interactions with the scissile phosphate in the ground state or for increased negative charge in the transition state. Moreover, the tremendous acceleration in the rate of hydrolysis of a methylphosphonate versus phosphodiester indicates that electrostatic repulsion of the water nucleophile is a major impediment to the latent nuclease activity of topoisomerase IB. Results Experimental Strategy to Probe the Influence of Charge and Polar Interactions of Topoisomerase at the Scissile Phosphodiester Vaccinia topoisomerase is a prototype of the type IB topoisomerase family, which includes eukaryotic nu-
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Figure 1. DNA Transesterification and Hydrolysis Reactions of Topoisomerase IB (A) The reaction pathway for transesterification at a DNA phosphodiester entails nucleophilic attack of tyrosine on the phosphorus to form a covalent DNA-(3⬘-PO4)-enzyme intermediate and a 5⬘-OH DNA strand. The reaction proceeds through a pentacoordinate phosphorane transition state. (B) The hypothetical reaction pathway for transesterification at a DNA methylphosphonate linkage. The methylphosphonate is uncharged in the ground state and has a nominal charge of ⫺1 in the proposed transition state. The reaction proceeds with inversion of stereochemical configuration at the chiral phosphorus center. (C) Attack by water on the covalent phosphodiester intermediate expels the tyrosine leaving group and yields a 3⬘-phosphate DNA strand. (D) Hydrolysis of the chiral covalent methylphosphonate intermediate generates an achiral 3⬘-methylphosphate DNA end.
clear and mitochondrial topoisomerases IB, other poxvirus topoisomerases, and poxvirus-like topoisomerases encoded by bacteria (Champoux, 2001; Zhang et al., 2001; Krogh and Shuman, 2002a). The vaccinia enzyme is distinguished by its compact size (314 amino acids) and its site specificity in DNA transesterification (Shuman, 1998). Vaccinia topoisomerase binds duplex DNA and nicks one strand at a pentapyrimidine target sequence, 5⬘-(T/C)CCTT↓ (Shuman and Prescott, 1990). The Tp↓ nucleotide (defined as the ⫹1 nucleotide) is linked to Tyr274 of the enzyme. Four conserved amino acid side chains found in all type IB topoisomerases (Arg130, Lys167, Arg223, and His265 in the vaccinia enzyme) are responsible for catalyzing the attack of the active site tyrosine nucleophile (Tyr274) on the scissile phosphodiester to form the covalent intermediate (Petersen and Shuman, 1997b; Wittschieben and Shuman, 1997; Cheng et al., 1997). Mutational, stereochemical, and structural data suggest
that Arg130 and His265 contact the nonbridging oxygens of the scissile phosphodiester and that these interactions serve to stabilize a proposed phosphorane transition state (Stivers et al., 2000; Krogh and Shuman, 2000b; Cheng et al., 1998; Redinbo et al., 1998) (Figure 1A). Lys167 and Arg130 comprise a proton relay that catalyzes the expulsion of the 5⬘-O of the leaving DNA strand (Krogh and Shuman, 2000a, 2002b). There is no requirement for, or participation of, a divalent cation in the transesterification reactions. The effects of DNA modifications on transesterification have provided insights to the topoisomerase reaction. For example, introduction of a 5⬘-phosphate/3⬘-OH nick in lieu of the scissile phosphodiester abolishes transesterification without affecting the noncovalent binding of topoisomerase to the nicked DNA (Cheng and Shuman, 1999). This result indicates that vaccinia topoisomerase is an obligate polynucleotidyl-3⬘-phosphotransferase and cannot transesterify to a 5⬘ phos-
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Figure 2. Sp Methylphosphonate Substitution at the Scissile Phosphodiester Converts Topoisomerase into an Endonuclease (A and B) The 18-mer/30-mer suicide substrate is illustrated at the bottom of the figure, and the cleavage site is indicated by an arrow. The scissile strand was 5⬘ 32P-labeled (indicated by an asterisk). Reaction mixtures contained 0.3 pmol DNA with an Sp methylphosphonate at the cleavage site and 2 pmol topoisomerase. Aliquots were withdrawn at the times specified and either (A) analyzed directly by SDS-PAGE or (B) digested with proteinase K and electrophoresed through a high-resolution TBE-urea sequencing gel. Autoradiographs of the gels are shown. The 5⬘ 32P-labeled reference oligonucleotides in the rightmost lanes of (B) were as follows: lane C, 5⬘-pCGTGTCGCCCTMePTATTCCCOH3⬘; lane M⬘, 5⬘-pCGTGTCGCMePCCTTp-3⬘; lane M, 5⬘-pCGTGTCGCCCTTp-3⬘.
phomonoester. Because a phosphodiester has a nominal net charge of ⫺1 in the ground state and ⫺2 in the proposed transition state (Figure 1A), whereas a 5⬘ phosphomonoester has a nominal charge of ⫺2 in the ground state (and would acquire a charge of ⫺3 in an associative transition state), it is possible that electrostatic repulsion of the attacking tyrosine nucleophile by the extra charge precludes a reaction with the 5⬘ monoester. Introduction of chiral phosphorothioates at the cleavage site elicits stereoselective reductions in the rate of transesterification (30-fold and 340-fold for the Sp and Rp diastereomers) without affecting the yield of the covalent intermediate (Stivers et al., 2000). The phosphorothioate modification imposes no change in net charge; however, the negative charge will localize largely on the sulfur, instead of distributing over the two nonbridging oxygens, as in a phosphodiester (Iyengar et al., 1984; Eckstein, 2000). The thio effects on cleavage rate are attributed to destabilization of the transition state, either via perturbation of the electrostatic environment or via steric hindrance imposed by the larger van der Waals radius of sulfur (1.85 A˚) compared to oxygen (1.44 A˚). Here, we use chiral methylphosphonate-substituted DNAs to probe the contributions of negative charge on the scissile phosphodiester and presumptive polar contacts of the enzyme with the nonbridging oxygens in the catalysis of the strand cleavage reaction. The methylphosphonate modification eliminates the negative charge on the phosphodiester in the ground state and would reduce the charge on the transition state
from ⫺2 to ⫺1 (Figure 1B). Also, the methyl group would not be able to engage in the hydrogen bonding interactions with arginine or histidine side chains that are invoked for the transition state. Sp Methylphosphonate Substitution at the Scissile Phosphodiester Converts Vaccinia Topoisomerase into a Nuclease We synthesized an 18-mer strand containing an Sp methylphosphonate at the CCCTTpA cleavage site. The methylphosphonate strand and an unmodified phosphodiester control strand were 5⬘ 32P-labeled and annealed to an unlabeled 30-mer complementary strand to form “suicide” substrates for vaccinia topoisomerase (Figure 2). Transesterification at the TpA linkage results in covalent attachment of a 5⬘ 32P-labeled 12-mer (5⬘pCGTGTCGCCCTTp-3⬘) to the enzyme via Tyr274. The unlabeled 6-mer 5⬘-OH leaving strand (5⬘-ATTCCC-3⬘) dissociates spontaneously from the protein-DNA complex and thereby drives the reaction toward the covalent state, so that the reaction can be treated kinetically as a unidirectional process (Stivers et al., 1994; Wittschieben and Shuman, 1997). Formation of the covalent adduct is detectable by SDS-PAGE analysis of the reaction products. The 32P-labeled DNA is transferred to the topoisomerase polypeptide to form an ⵑ40 kDa protein-DNA adduct that is well resolved from the DNA, which migrates near the dye front (Figure 2A). The reaction of topoisomerase with the unmodified phosphodiester substrate attained an endpoint of 93% covalent adduct within 20 s; from the extent of adduct formation at 5 s,
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we calculated a cleavage rate constant of 0.4 s⫺1. Note that the second order process of noncovalent binding of topoisomerase to the DNA target site is fast and saturating under the conditions used (e.g., the observed cleavage rate constant does not increase when the topoisomerase concentration is increased 2-fold), thus justifying the estimation of a pseudo-first order rate constant from the data. Analysis of product formation during a reaction of topoisomerase with the Sp methylphosphonate substrate revealed a transient accumulation of the covalent topoisomerase-DNA complex, which peaked at 3–5 min and declined steadily thereafter (Figure 2A). Concomitant with the disappearance of the topoisomerase-DNA adduct, there accumulated a novel 32P-labeled species that migrated at the solute front and was clearly separated from the input 18-mer oligonucleotide (Figure 2A). To identify the novel radiolabeled species, the reaction products were digested with proteinase K and then analyzed by electrophoresis through a 17% polyacrylamide/TBE/7 M urea gel that resolves DNAs on the basis of chain length. As shown in Figure 2B, the novel labeled product corresponds to a free 32P-labeled 12-mer DNA. The kinetics of accumulation of the 12-mer strand paralleled the appearance of the rapidly migrating product detected by SDS-PAGE. The analysis in Figure 2B also revealed the accumulation and disappearance of a cluster of radiolabeled products of proteolysis of the covalent intermediate, migrating between the 18-mer and 12-mer strands. This cluster (indicated by arrowheads in Figure 2B) consists of the 5⬘ 32P-labeled 12-mer strand linked to heterogeneous short peptides. Detection of the cluster in this gel system depended on proteinase K treatment, because the intact topoisomerase-DNA adduct does not enter the gel. The abundance of the labeled free 12-mer was not affected by omission of the proteolysis step (not shown). The kinetic profile of the formation of the covalent and free products (Figure 3A) is consistent with a precursorproduct relationship, whereby the topoisomerase first reacts with the Sp methylphosphonate substrate to form a DNA-(3⬘-methylphosphonate)-tyrosyl linkage, which is then hydrolyzed to liberate the free 12-mer DNA. The hydrolysis reaction is both efficient (95% of the input 18-mer strand being converted to the free 12-mer) and relatively rapid, being complete in 1 hr and attaining 50% of the endpoint in 10 min. The data in Figure 3A were fit by using the CKS kinetic simulation program (version 1.0; IBM) to a unidirectional two-step reaction mechanism with transesterification and hydrolysis rate constants of 0.0018 s⫺1 and 0.006 s⫺1, respectively (Figure 4). The apparent rate of transesterification of topoisomerase to the Sp methylphosphonate linkage was about 220-fold slower than the rate of covalent adduct formation at a phosphodiester (Figure 4). The reduced rate of topoisomerase transesterification to the Sp methylphosphonate DNA did not reflect interference with the noncovalent binding step, insofar as the observed rates of transesterification and hydrolysis did not increase when the concentration of topoisomerase was doubled (not shown).
Figure 3. Kinetic Profile of the Reaction of Topoisomerase with Methylphosphonate DNAs The abundance of the covalent methylphosphonate intermediate and the free 12-mer DNA (expressed as the percent of total 32P label in each species) is plotted as a function of time. (A) Sp methylphosphonate DNA. (B) Rp methylphosphonate DNA.
Different Effects of Sp and Rp Methylphosphonate Diastereomers We next analyzed the reaction of vaccinia topoisomerase with a 5⬘ 32P-labeled substrate containing an Rp methylphosphonate modification at the CCCTTpA cleavage site. Product analysis revealed a time-dependent accumulation of the rapidly migrating free DNA species at the solvent front, concomitant with the decay of the more slowly migrating substrate strand (Figure 5A). Trace levels of the covalent topoisomerase-DNA complex were seen from 2 to 120 min and declined at later times (see the top panel insert in Figure 5A). The covalent adduct to the Rp substrate comprised no more than ⵑ0.6% of the 32P label during this interval (compared to a peak value of 17% covalent adduct for the Sp substrate). Analysis of proteinase K-digested reaction products demonstrated the steady accumulation of a free 12-mer hydrolysis product (Figure 5B); in this exposure, the cluster of covalent 12-mer peptide adducts was not detectable.
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Figure 4. Rate Constants for Transesterification and Hydrolysis of Phosphodiester and Methylphosphonate DNAs by Wild-Type Vaccinia Topoisomerase and the H265A Mutant See the text for discussion. The structures of a standard DNA phosphodiester and the Sp and Rp methylphosphonate (MeP) diastereomers are illustrated. nd; not determined.
The kinetic profile of the hydrolysis of the Rp methylphosphonate is shown in Figure 3B. The extent of hydrolysis of the Rp substrate (ⵑ95%) was similar to that of the Sp diastereomer; however, the rate of Rp hydrolysis was obviously slower than the Sp reaction. Hydrolysis of the Rp DNA was complete in 8 hr and reached 50% of the endpoint in 2 hr (compared to 10 min for Sp DNA). The difference is attributable to a slower rate of transesterification by topoisomerase to the Rp methylphosphonate than to the Sp substrate. We did not detect a lag in the formation of the free 12-mer from the Rp DNA; this finding suggests a kinetic mechanism in which the rate constant for transesterification is at least 10fold slower than the rate of hydrolysis of the covalent intermediate. From a kinetic simulation, we estimated transesterification and hydrolysis rate constants of 0.00011 s⫺1 and 0.009 s⫺1, respectively (Figure 4). The rates of transesterification and hydrolysis on the Rp methylphosphonate DNA did not increase when the concentration of topoisomerase was doubled (not shown). Comparison of the rate constants for the Sp and Rp methylphosphonate substrates highlights a significant stereochemical effect on the transesterification step (Sp/Rp ⫽ 16), but not on the hydrolysis step. Because the transesterification reaction occurs with inversion of configuration at the phosphorus being attacked, the substrates for hydrolysis, which we presume to be the covalent DNA-(3⬘ methylphosphonate)-enzyme complex, will be of opposite chirality relative to the respective starting DNAs. Thus, hydrolysis of the Sp methylphosphonate DNA would entail attack of water on the stereochemically inverted diastereomer of the covalent intermediate, assuming that the covalent complex is an obligate intermediate along the hydrolysis reaction pathway. Hydrolysis Reaction Mechanism and Characterization of the Product If the methylphosphonate-dependent hydrolysis reactions occur via an obligate covalent intermediate, then
the active site tyrosine nucleophile (Tyr274) should be essential for the overall reactions with the Sp and Rp methylphosphonate substrates. On the other hand, if hydrolysis can occur via direct attack of water on the DNA methylphosphonate, then the tyrosine nucleophile might be dispensable. We gave serious consideration to the second pathway in light of studies of FLP recombinase showing that FLP mutants lacking the active site tyrosine, though inert in forming the covalent proteinDNA adduct, are nonetheless capable of hydrolyzing DNA in the presence of a strong nucleophile via direct attack on the scissile phosphodiester (Lee and Jayaram, 1993). Here, we found that the Y274A mutant of vaccinia topoisomerase was incapable of forming a free 12-mer strand when reacted with either the Sp or Rp methylphosphonate substrates during incubations of up to 24 hr (not shown), arguing that transesterification by Tyr274 at the methylphosphonate is a prerequisite for the observed hydrolysis reactions. In principle, the hydrolysis of the DNA-(3⬘-methylphosphotyrosyl)-enzyme linkage could yield either a DNA3⬘-methylphosphate strand plus the native tyrosyl enzyme or a DNA-3⬘-OH strand plus a phosphotyrosyl enzyme. The nature of the DNA 3⬘ terminus was characterized by electrophoresis of the 5⬘ 32P-labeled 12-mer hydrolysis product in parallel with a 5⬘ 32P-labeled 12mer of identical sequence that contained a 3⬘-phosphate end. The latter species (M in Figures 2B and 5B) was generated by peroxidolysis of the covalent adduct formed by topoisomerase on a standard phosphodiester substrate (Krogh and Shuman, 2000a). The methylphosphonate hydrolysis reaction products migrated just slightly slower than the 3⬘ phosphate-terminated 12-mer (Figures 2B and 5B). We surmised that this small mobility change reflected the difference in charge on the 3⬘ phosphate (net charge of ⫺2 at pH 8) versus a 3⬘ methylphosphate (net charge of ⫺1). This idea was supported by the finding that a 5⬘ 32P-labeled 12-mer of identical sequence containing a 3⬘-phosphate terminus and single internal methylphosphonate linkage (lane M in Figure 2B) mi-
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Figure 5. Reaction of Topoisomerase at an Rp Methylphosphonate Linkage (A and B) Reaction mixtures contained 0.3 pmol DNA with an Rp methylphosphonate at the cleavage site and 2 pmol topoisomerase. Aliquots were withdrawn at the times specified and either (A) analyzed directly by SDSPAGE or (B) digested with proteinase K and electrophoresed through a high-resolution DNA sequencing-type gel. Autoradiographs of the gels are shown. (A) includes a prolonged exposure of the SDS gel, revealing trace levels of the covalent intermediate. In (B), lane M contains the 5⬘-radiolabeled reference oligonucleotide pCGTGTCGCCCTTp.
grated identically to the methylphosphonate hydrolysis product (note that they have the same net charge on the 12-mer) and slightly slower than the unmodified 3⬘phosphate strand (lane M). The hydrolysis products of the Rp and Sp methylphosphonate DNAs migrated faster than a 5⬘ 32P-labeled 12-mer of identical sequence containing a 3⬘ hydroxyl terminus; this finding provides further evidence that the hydrolysis reaction yielded a 3⬘ methylphosphonate terminus (Figure 6, lane M). To confirm the assignments of the 3⬘ ends, we treated the Rp and Sp hydrolysis reaction products and the peroxidolysis reaction product (containing a 3⬘-phosphate terminus) with the enzyme T4 polynucleotide 3⬘ phosphatase. The peroxidolysis product was thereby converted quantitatively into a slower species that comigrated with the 3⬘-OH DNA standard. In contrast, neither of the methylphosphonate hydrolysis reaction products was affected by treatment with 3⬘ phosphatase, which attests to the fact that they have modified 3⬘-methylphosphate termini that are not susceptible to hydrolysis by the T4 enzyme.
Methylphosphonates Stimulate the Rate of Topoisomerase-Catalyzed Hydrolysis The rates of hydrolysis of the methylphosphonate-substituted covalent intermediates (0.006–0.009 s⫺1) are considerably faster than expected from previous estimates of the rate of topoisomerase-catalyzed hydrolysis
of the unmodified covalent phosphodiester intermediate under optimal alkaline pH conditions (Krogh and Shuman, 2000a). In order to directly evaluate the magnitude of the gain-of-function in hydrolysis elicited by the methylphosphonates, we determined the rate of hydrolysis of an unmodified suicide intermediate, comprising 95% of the total labeled DNA at 1 min after initiation of the reaction, by taking time points at 1-day intervals over a 7-day incubation a pH 7.5. The free 12-mer product accumulated progressively to an extent of 10% of the total labeled DNA after 7 days (data not shown). From the initial rate, we calculated a rate constant of 2.2 ⫻ 10⫺7 s⫺1 for hydrolysis of the DNA-3⬘-phosphotyrosyl linkage. Thus, replacement of a nonbridging oxygen on the covalent adduct by a methyl group accelerated the hydrolysis reaction by a factor of ⵑ30,000 (Figure 4). “Biochemical Epistasis” between His265 and the Sp Methyl Group His265 is an essential component of the topoisomerase active site that functions in transition state stabilization and not as a general acid-base catalyst (Krogh and Shuman, 2000a; Stivers et al., 2001). The fact that His265 could be replaced by either asparagine or glutamine suggested that this side chain contributes a neutral hydrogen bond to one of the nonbridging oxygens in the transition state (Petersen and Shuman, 1997b). The 200fold rate decrement in transesterification imposed by the Sp methyl group was quantitatively similar to the
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Figure 6. The Hydrolysis Reaction Product Is a DNA-3⬘-Methylphosphate The free 12-mer products of topoisomerase-catalyzed peroxidolysis of the covalent phosphodiester intermediate (control) and topo-catalyzed hydrolysis of the Rp and Sp methylphosphonate substrates were analyzed by denaturing PAGE either with (⫹) or without (⫺) prior treatment with T4 polynucleotide 3⬘-phosphatase. An autoradiograph of the gel is shown. The duration of the individual peroxidolysis or hydrolysis reactions was varied so as to attain near-quantitative conversion of the input 32P-labeled 18-mer strand (shown in the leftmost lane) to free 12-mer. The DNAs were recovered by phenol extraction and ethanol precipitation. Aliquots were incubated for 20 min at 37⬚C in phosphatase reaction mixtures (25 l) containing 100 mM imidazole (pH 6.0), 10 mM MgCl2, 10 mM -mercaptoethanol, 0.1 mg/ml BSA, and 10 ng purified recombinant T4 polynucleotide 3⬘-phosphatase. The digestion products were recovered by phenol extraction and ethanol precipitation. Lane M contained the 5⬘-radiolabeled reference oligonucleotide pCGTGTCGCCCTTOH.
effect of replacing catalytic residue His265 with alanine, i.e., the rate of single-turnover transesterification by the H265A mutant enzyme on the unmodified suicide substrate was 0.0018 s⫺1 (Figure 4). We therefore speculated that in the transition state, His265 forms a hydrogen bond to the oxygen corresponding to the Sp methyl. We tested this model by examining the reaction of the H265A mutant with the Rp and Sp methylphosphonatemodified DNAs. A prediction of the model is that if catalysis by His265 entails contact only with the pro Sp oxygen, then elimination of the His side chain should have little or no impact on the reaction of topoisomerase with the Sp methylphosphonate-substituted DNA. A corollary prediction is that the reaction of the Rp methylphosphonate substrate with topoisomerase should be slowed significantly by the loss of the stabilizing contact of His265 to the lone nonbridging oxygen. This reasoning echoes genetic epistasis analysis, whereby the effects of mutations in two genes in the same pathway are not exacerbated when combined, but mutations affecting parallel pathways are additive when combined. The extent and kinetic profile of the reaction of H265A with the Sp methylphosphonate DNA (Figure 7A) was nearly identical to that of wild-type topoisomerase on the same substrate (Figure 3A). The covalent intermedi-
Figure 7. Reaction of Topoisomerase Mutant H265A with Methylphosphonate DNAs (A and B) Reaction mixtures contained 0.3 pmol Sp or Rp methylphosphonate DNA and 2 pmol H265A topoisomerase. Aliquots were withdrawn at the times specified, digested with proteinase K, and then analyzed by TBE-urea PAGE. The product distributions are plotted as a function of time for the (A) Sp and (B) Rp substrates. No covalent intermediate was detected in the reaction of H265A with the Rp methylphosphonate substrate.
ate accumulated transiently at 2–5 min and decayed thereafter as free 12-mer was formed. From a kinetic simulation, we estimated transesterification and hydrolysis rate constants of 0.0024 s⫺1 and 0.0085 s⫺1, respectively (Figure 4). The H265A mutation had no deleterious effect on the rate of formation of the covalent complex on the Sp methylphosphonate DNA or its hydrolysis (Figure 4). The reaction profile of H265A on the Rp methylphosphonate DNA (Figure 7B) showed significant slowing of the composite transesterification/hydrolysis reaction compared to that of the wild-type enzyme on the same DNA (Figure 3B). The free 12-mer accumulated steadily over a 6-day incubation to an extent of 23% of the input-labeled scissile strand. We were unable to detect any accumulation of covalent intermediate at any of the time points analyzed (at a limit of detection of 0.2% of total labeled DNA). This lack of accumulation signified that the overall reaction rate was limited by the DNA transesterification step and that the rate of the hydrolysis step was much faster than the rate of transes-
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terification. Thus, we fit the data to a single-exponential decay function and calculated a rate constant of 6 ⫻ 10⫺7 for formation of the 12-mer. This value can be taken as a reasonable approximation of the rate of transesterification of H265A at the Rp methylphosphonate linkage. Thus, the His265A mutation reduced kcl on the Rp DNA by a factor of 200 compared to that of wild-type topoisomerase (Figure 4). The magnitude of this decrement was nearly identical to that elicited by the H265A mutation on transesterification at an unmodified phosphodiester. These results provide evidence that the essential histidine of topoisomerase IB assists in catalysis of transesterification exclusively via contacts to the pro Sp oxygen of the scissile phosphodiester. They also illustrate the power of “biochemical epistasis” tests and chiral methylphosphonates to illuminate functionally relevant protein-DNA contacts at atomic resolution in the absence of an atomic structure. Discussion Chiral methylphosphonates have been used previously to test the effects of backbone charge neutralization on protein-nucleic acid binding interactions (Noble et al., 1984; Pritchard et al., 1994; Dertinger and Uhlenbeck, 2001), but they have not been applied widely to the analysis of phosphoryl transfer enzymes (Rosati et al., 2002). Topoisomerase IB is an excellent case study for the utility of the methylphosphonates to dissect an enzyme mechanism, insofar as the transesterification chemistry is independent of a metal cofactor and catalysis is presumed to entail stabilizing contacts between amino acids of the enzyme and the nonbridging phosphate oxygens of the proposed transition state. Here, we have shown that chiral methylphosphonate substitutions at the cleavage site elicit two highly instructive effects on vaccinia virus topoisomerase IB. First, the Sp and Rp methylphosphonate diastereomers slow the rate of DNA transesterification by factors of 220 and 3600, respectively. Second, they unmask a latent and highly potent hydrolytic activity, effectively converting the topoisomerase into a nuclease. The stereo-specific methylphosphonate interference effects on transesterification (Sp/Rp ⫽ 16) underscore the distinct contributions of electrostatic and/or hydrogen bonding interactions with the two different nonbridging phosphate oxygens. A simple interpretation is that each oxygen interacts with a different partner on the enzyme. The rate decrement associated with the Sp methyl group is quantitatively similar to the effect of replacing catalytic residue His265 with alanine (Petersen and Shuman, 1997b), and we have presented functional evidence that the histidine stabilizes the transition state by donating a hydrogen bond to the oxygen corresponding to the Sp methyl. This view is in keeping with the inferences of Stivers et al. (2000) concerning the contacts of His265 with the nonbridging oxygens, based on the effects of chiral phosphorothioate modifications at the cleavage site. (To avoid confusion in comparing the two studies, it must be kept in mind that the nomenclature for the Rp and Sp configurations of methylphosphonates is opposite to that of the chiral phosphorothioates, such that an Rp methylphosphonate is substituted at
the same oxygen as the Sp phosphorothioate and the Sp methylphosphonate is substituted at the same oxygen as an Rp phosphorothioate.) The fact that transesterification occurs as rapidly as it does on the Sp methylphosphonate DNA attests to the lack of a stringent requirement for ionic interactions between topoisomerase IB and the nonbridging phosphate oxygens in the ground state or for the obligate presence of two negative charges on the transition state. This contrasts with the recent report that DNA cleavage by the EcoRI restriction endonuclease is abolished absolutely by Rp or Sp methylphosphonate modification at the scissile phosphodiester (Rosati et al., 2002). One of the methylphosphonate diastereomers strongly affected the binding of EcoRI to its target site, but the other diastereomer did not. In the present study, the reduced rates of topoisomerase transesterification to the methylphosphonate-modified DNAs do not reflect interference with noncovalent binding, insofar as the reaction rates did not increase when the concentration of topoisomerase was doubled. The activation of the latent nuclease of vaccinia topoisomerase by methylphosphonates is the result of a 30,000- to 40,000-fold increase in the rate of self-catalyzed hydrolysis of the covalent methylphosphonate intermediate vis a vis the tyrosyl-DNA phosphodiester (Figures 1C and 1D). Whereas the phosphodiester intermediate has a half-life of 36 days at physiological pH, the methylphosphonate intermediate has a half-life of less than 2 min. This prodigious gain-of-function was independent of the stereochemical configuration of the methylphosphonate intermediate and is therefore likely to be attributable to altered electrostatics. Note that the accelerated hydrolysis reaction was not caused by an inherent chemical lability of the tyrosine-methylphosphonate-DNA linkage, insofar as the covalent intermediate formed by the wild-type or H256A topoisomerases at early times on the Sp methylphosphonate substrate (Figures 3A and 7A) was stable for at least 12 hr at room temperature after the topoisomerase was denatured with SDS. Further evidence that hydrolysis of the covalent methylphosphonate intermediate was catalyzed directly by the topoisomerase active site stems from our finding that a mutation of another catalytic amino acid (Arg223) elicited a 400-fold decrement in khydrol on the Rp methylphosphonate substrate (L.T. and S.S., unpublished data). A plausible explanation for the gain-of-function is that hydrolysis is normally strongly suppressed by electrostatic repulsion of the nucleophilic water and that neutralization of the negative charge by methyl substitution for either the pro Rp or pro Sp oxygens removes this barrier to attack by water on the covalent intermediate. It is truly remarkable that the rate of hydrolysis of the methylphosphonate intermediates by wild-type and H265A topoisomerase (0.006–0.009 s⫺1) was only two orders of magnitude slower than the normal religation step of the topoisomerase catalytic cycle (krel ⫽ 1 s⫺1), which entails attack on the covalent intermediate by a DNA 5⬘-OH that is optimally aligned at the active site by virtue of base pairing of the acceptor polynucleotide to the nonscissile strand. Note that the rate of hydrolysis of the methylphosphonate intermediates was as fast or
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faster than DNA religation across a 1 nucleotide gap, where krel ⫽ 0.005 s⫺1 (Sekiguchi et al., 1997). Previous studies showed that the H265A mutation slowed the rate of self-catalyzed hydrolysis of the covalent phosphodiester intermediate at alkaline pH by nearly two orders of magnitude (Krogh and Shuman, 2000a). Given that the observed khydrol for the wild-type topoisomerase-DNA phosphodiester intermediate at pH 7.5 was 2.2 ⫻ 10⫺7 s⫺1, a conservative estimate is that the rate of hydrolysis by the H265A mutant under the present reaction conditions would be ⬍10⫺8 s⫺1 (Figure 4). Comparison to the observed khydrol of the Sp-modified 18-mer substrate by the H265A mutant (0.0085 s⫺1) highlights a gain of hydrolytic function for the mutant enzyme of about six orders of magnitude. In summary, methylphosphonates expose the Achilles’ heel of topoisomerase IB as a guardian of genomic integrity. The topoisomerase is capable of vigorous hydrolytic activity, but reaction of the covalent intermediate with water is suppressed by virtue of electrostatic repulsion at the active site. Simple neutralization of the charge on the scissile phosphate of the covalent intermediate elicits a quantitative gain-of-function that, to our knowledge, is unprecedented in an enzymatic phosphoryl transfer reaction. Our results focus attention on repulsion of the attacking nucleophile as a potentially limiting factor in other enzymatic reactions at phosphates (especially metal-independent reactions), and they underscore the power of chiral methylphosphonates to address this question. Experimental Procedures DNA Substrates Oligonucleotides containing single chiral methylphosphonates were prepared by incorporating isomerically pure Sp and Rp dinucleotide methylphosphonate 5⬘-TpA cassettes during standard phosphoramidite-based oligonucleotide synthesis protocols. Synthesis and purification of the TpA methylphosphonate cassettes were performed essentially as described (Reynolds et al., 1996). The methylphosphonate dinucleotides were ⬎95% diastereochemically pure, as determined by analytical HPLC with peak integration and 31P NMR spectroscopic analysis. Detailed methods for chemical synthesis will be reported separately. The CCCTT-containing scissile strands were 5⬘ 32P-labeled by enzymatic phosphorylation in the presence of [␥-32P] ATP and T4 polynucleotide kinase. The 18-mer labeled oligonucleotides were gel purified and annealed to an unlabeled 30-mer oligonucleotide (containing a 5⬘-PO4 terminus introduced during chemical synthesis) at a 1:4 molar ratio of 18mer:30-mer. Vaccinia Topoisomerase Recombinant vaccinia topoisomerase was produced in E. coli BL21 by infection with bacteriophage CE6 and then purified to apparent homogeneity from the soluble bacterial lysate by phosphocellulose and Source S-15 chromatography steps. Protein concentration was determined by using the dye binding method (Biorad) with bovine serum albumin as the standard. DNA Transesterification and Hydrolysis Reaction mixtures containing (per 20 l) 50 mM Tris-HCl (pH 7.5), 0.3 pmol 18-mer/30-mer DNA, and 75 ng vaccinia topoisomerase were incubated at 37⬚C. Aliquots (20 l) were withdrawn at the times specified and quenched immediately with SDS (1% final concentration). The products were analyzed by SDS-PAGE. Duplicate aliquots were quenched with SDS and were digested for 2 hr at 37⬚C with 10 g proteinase K. The mixtures were adjusted to 47% formamide, heat denatured, and electrophoresed through a 20% denaturing
polyacrylamide gel containing 7 M urea in TBE (90 mM Tris-borate, 2.5 mM EDTA). The reaction products were visualized by autoradiography and were quantified by scanning the gels with a Fujifilm BAS2500 phosphorimager. Acknowledgments This work was supported by National Institutes of Health grants GM46330 (S.S.) and CA78415 (S.M.H.). Received: March 11, 2003 Revised: April 14, 2003 Accepted: June 11, 2003 Published: July 24, 2003 References Burgin, A.B., Huizenga, B.H., and Nash, H.A. (1995). A novel suicide substrate for DNA topoisomerases and site-specific recombinases. Nucleic Acids Res. 15, 2973–2979. Champoux, J.J. (2001). DNA topoisomerases: structure, function, and mechanism. Annu. Rev. Biochem. 70, 369–413. Cheng, C., and Shuman, S. (1999). Site-specific transesterification by vaccinia topoisomerase: role of specific phosphates and nucleosides. Biochemistry 38, 16599–16612. Cheng, C., Wang, L.K., Sekiguchi, J., and Shuman, S. (1997). Mutational analysis of 39 residues of vaccinia DNA topoisomerase identifies Lys-220, Arg-223, and Asn-228 as important for covalent catalysis. J. Biol. Chem. 272, 8263–8269. Cheng, C., Kussie, P., Pavletich, N., and Shuman, S. (1998). Conservation of structure and mechanism between eukaryotic topoisomerase I and site-specific recombinases. Cell 92, 841–850. Christiansen, K., Knudsen, B.R., and Westergaard, O. (1994). The covalent eukaryotic topoisomerase I–DNA intermediate catalyzes pH-dependent hydrolysis and alcohololysis. J. Biol. Chem. 269, 11367–11373. Dertinger, D., and Uhlenbeck, O.C. (2001). Evaluation of methylphosphonates as analogs for detecting phosphate contacts in RNAprotein complexes. RNA 7, 622–631. Eckstein, F. (2000). Phosphorothioate oligonucleotides: what is their origin and what is unique about them? Antisense Nucleic Acid Drug Dev. 10, 117–121. Henningfield, K.A., Arslan, T., and Hecht, S.M. (1996). Alteration of DNA primary structure by DNA topoisomerase I: isolation of the covalent topoisomerase I-DNA binary complex in enzymatically competent form. J. Am. Chem. Soc. 118, 11701–11714. Hsiang, Y.H., Hertzberg, R., Hecht, S., and Liu, L.F. (1985). Camptothecin induces protein-linked DNA breaks via mammalian DNA topoisomerase I. J. Biol. Chem. 260, 14873–14878. Iyengar, R., Eckstein, F., and Frey, P.A. (1984). Phosphorus-oxygen bond order in adenosine 5⬘-O-phosphorothioate dianion. J. Am. Chem. Soc. 106, 8309–8310. Krogh, B.O., and Shuman, S. (2000a). Catalytic mechanism of DNA topoisomerase IB. Mol. Cell 5, 1035–1041. Krogh, B.O., and Shuman, S. (2000b). DNA strand transfer catalyzed by vaccinia topoisomerase: peroxidolysis and hydroxylaminolysis of the covalent protein-DNA intermediate. Biochemistry 39, 6422– 6432. Krogh, B.O., and Shuman, S. (2002a). A poxvirus-like type IB topoisomerase family in bacteria. Proc. Natl. Acad. Sci. USA 99, 1853– 1858. Krogh, B.O., and Shuman, S. (2002b). Proton relay mechanism of general acid catalysis by DNA topoisomerase IB. J. Biol. Chem. 277, 5711–5714. Krogh, B.O., Cheng, C., Burgin, A., and Shuman, S. (1999). Melanoplus sanguinipes entomopoxvirus DNA topoisomerase: site-specific DNA transesterification and effects of 5⬘-bridging phosphorothiolates. Virology 264, 441–451. Lanza, A., Tornaletti, S., Rodolfo, C., Scanavini, C., and Pedrini, A.M. (1996). Human DNA topoisomerase I-mediated cleavages stimu-
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lated by ultraviolet light-induced DNA damage. J. Biol. Chem. 271, 6978–6986. Lee, J., and Jayaram, M. (1993). Mechanism of site-specific recombination: logic of assembling recombinase catalytic site from fractional active sites. J. Biol. Chem. 268, 17564–17579. Noble, S.A., Fisher, E.F., and Caruthers, M.H. (1984). Methylphosphonates as probes of protein-nucleic acid interactions. Nucleic Acids Res. 12, 3387–3404. Petersen, B.Ø., and Shuman, S. (1997a). DNA strand transfer reactions catalyzed by vaccinia topoisomerase: hydrolysis and glycerololysis of the covalent protein-DNA intermediate. Nucleic Acids Res. 25, 2091–2097. Petersen, B.Ø., and Shuman, S. (1997b). Histidine-265 is important for covalent catalysis by vaccinia topoisomerase and is conserved in all eukaryotic type I enzymes. J. Biol. Chem. 272, 3891–3896. Pommier, Y., Kohlhagen, G., Pourquier, P., Sayer, J.M., Kroth, H., and Jerina, D.M. (2000). Benzo[a]pyrene diol epoxide adducts in DNA are potent suppressors of a normal topoisomerase I cleavage site and powerful inducers of other topoisomerase I cleavages. Proc. Natl. Acad. Sci. USA 97, 2040–2045. Pommier, Y., Kohlhagen, G., Laco, G.S., Kroth, H., Sayer, J.M., and Jerina, D.M. (2002). Differential effects on human topoisomerase I by minor groove and intercalated deoxyguanine adducts derived from two polycyclic aromatic hydrocarbon diol epoxides at or near a normal cleavage site. J. Biol. Chem. 277, 13666–13672. Pourquier, P., Ueng, L., Kohlhagen, G., Mazumder, A., Gupta, M., Kohn, K.W., and Pommier, Y. (1997). Effects of uracil incorporation, DNA mismatches, and abasic sites on DNA cleavage and religation activities of mammalian topoisomerase I. J. Biol. Chem. 272, 7792– 7796. Pourquier, P., Bjornsti, M.A., and Pommier, Y. (1998). Induction of topoisomerase I cleavage complexes by the vinyl chloride adduct 1,N6-ethenoadenine. J. Biol. Chem. 273, 27245–27249. Pourquier, P., Ueng, L., Fertala, J., Wang, D., Park, H., Essigmann, J.M., Bjornsti, M.A., and Pommier, Y. (1999). Induction of reversible complexes between eukaryotic topoisomerase I and DNA containing oxidative base damages. J. Biol. Chem. 274, 8516–8523. Pritchard, C.E., Grasby, J.A., Hamy, F., Zacharek, A.M., Singh, M., Karn, J., and Gait, M.J. (1994). Methylphosphonate mapping of phosphate contacts critical for RNA recognition by the human immunodeficiency virus Tat and Rev proteins. Nucleic Acids Res. 22, 2592–2600. Redinbo, M.R., Stewart, L., Kuhn, P., Champoux, J.J., and Hol, W.G.J. (1998). Crystal structures of human topoisomerase I in covalent and noncovalent complexes with DNA. Science 279, 1504–1513. Reynolds, M.A., Hogrefe, R.I., Jaeger, J.A., Schwartz, D.A., Riley, T.A., Marvin, W.B., Daily, W.J., Vaghefi, M.M., Beck, T.A., Knowles, S.K., et al. (1996). Synthesis and thermodynamics of oligonucleotides containing chirally pure Rp methylphosphonate linkages. Nucleic Acids Res. 24, 4584–4591. Rosati, O., Srivastava, T.K., Katti, S.B., and Alves, J. (2002). Importance of phosphate contacts for sequence recognition by EcoRI restriction enzyme. Biochem. Biophys. Res. Commun. 295, 198–205. Sekiguchi, J., Cheng, C., and Shuman, S. (1997). Kinetic analysis of DNA and RNA strand transfer reactions catalyzed by vaccinia topoisomerase. J. Biol. Chem. 272, 15721–15728. Shuman, S. (1998). Vaccinia virus DNA topoisomerase: a model eukaryotic type IB enzyme. Biochim. Biophys. Acta 1400, 321–337. Shuman, S., and Prescott, J. (1990). Specific DNA cleavage and binding by vaccinia virus DNA topoisomerase I. J. Biol. Chem. 265, 17826–17836. Staker, B.L., Hjerrild, K., Feese, M.D., Behnke, C.A., Burgin, A.B., and Stewart, L. (2002). The mechanism of topoisomerase I poisoning by a camptothecin analog. Proc. Natl. Acad. Sci. USA 99, 15387– 15392. Stivers, J.T., Shuman, S., and Mildvan, A.S. (1994). Vaccinia DNA topoisomerase I: single-turnover and steady-state kinetic analysis of the DNA strand cleavage and ligation reactions. Biochemistry 33, 327–339.
Stivers, J.T., Jagadeesh, G.J., Nawrot, B., Stec, W.J., and Shuman, S. (2000). Stereochemical outcome and kinetic effects of Rp and Sp phosphorothioate substitutions at the cleavage site of vaccinia type I DNA topoisomerase. Biochemistry 39, 5561–5572. Wittschieben, J., and Shuman, S. (1997). Mechanism of DNA transesterification by vaccinia topoisomerase: catalytic contributions of essential residues Arg-130, Gly-132, Tyr-136, and Lys-167. Nucleic Acids Res. 25, 3001–3008. Zhang, H., Barcelo, J.M., Lee, B., Kohlhagen, G., Zimonjic, D.B., Popescu, N.C., and Pommier, Y. (2001). Human mitochondrial topoisomerase I. Proc. Natl. Acad. Sci. USA 98, 10608–10613.
Guarding the Genome: Electrostatic Repulsion of Water by DNA Suppresses a Potent Nuclease Activity of Topoisomerase IB Ligeng Tian,1 Christopher D. Claeboe,2 Sidney M. Hecht,2 and Stewart Shuman1,* 1 Molecular Biology Program Sloan-Kettering Institute New York, New York 10021 2 Departments of Chemistry and Biology University of Virginia Charlottesville, Virginia 22901
Summary Type IB topoisomerases cleave and rejoin DNA strands through a stable covalent DNA-(3ⴕ-phosphotyrosyl)-enzyme intermediate. The stability of the intermediate is a two-edged sword; it preserves genome integrity during supercoil relaxation, but it also reinforces the toxicity of drugs and lesions that interfere with the DNA rejoining step. Here, we identify a key determinant of the stability of the complex by showing that introduction of an Sp or Rp methylphosphonate linkage at the cleavage site transforms topoisomerase IB into a potent endonuclease. The nuclease reaction entails formation and surprisingly rapid hydrolysis of a covalent enzyme-DNA methylphosphonate intermediate. The ⵑ30,000-fold acceleration in the rate of hydrolysis of a methylphosphonate versus phosphodiester suggests that repulsion of water by the DNA phosphate anion suppresses the latent nuclease function of topoisomerase IB. These findings expose an Achilles’ heel of topoisomerases as guardians of the genome, and they have broad implications for understanding enzymatic phosphoryl transfer. Introduction Type IB topoisomerases alter DNA topology via a multistep reaction pathway entailing noncovalent binding of the enzyme to duplex DNA, cleavage of one DNA strand with formation of a covalent DNA-(3⬘-phosphotyrosyl)-protein intermediate, strand passage, and strand religation (Champoux, 2001; Shuman, 1998). The topoisomerase-DNA intermediate is extraordinarily stable at neutral pH. For example, vaccinia topoisomerase remains covalently bound to DNA for many days after “suicide cleavage” and remains fully competent to transfer the covalently held strand once it is provided with a 5⬘-OH acceptor DNA. The tyrosine-phosphodiester bond resists hydrolysis, even though the active site is accessible to solvent (Petersen and Shuman, 1997a; Krogh and Shuman, 2000b). The stability of the topoisomerase-DNA intermediate can be either a virtue or an Achilles’ heel, depending on the biological context. During normal topoisomerase function, which entails reiterative cleavage-ligation cycles at many sites in genomic DNA, it is important that the enzyme not relinquish its covalent hold on the 3⬘ *Correspondence: [email protected]
end of the nicked strand. To do so would abort the reaction and generate nicks that could be converted to lethal DNA breaks. In other situations, topoisomerase IB can become trapped in the covalent state, either by DNA lesions located at or 3⬘ to a cleavage site or by exposure to therapeutic drugs such as camptothecin (Burgin et al., 1995; Krogh et al., 1999; Lanza et al., 1996; Henningfield et al., 1996; Pourquier et al., 1997, 1998, 1999; Pommier et al., 2000, 2002; Hsiang et al., 1985; Staker et al., 2002). Such lesions and drugs selectively preclude the religation step by perturbing the position or reactivity of the DNA 5⬘-OH nucleophile with the DNA(3⬘-phosphotyrosyl)-topoisomerase complex. The covalent protein-DNA complex is itself a toxic lesion, and the toxicity is enforced by reluctance of the complex to undergo hydrolysis. In this setting, a nuclease activity associated with topoisomerase IB might be advantageous, allowing removal of topoisomerase from the DNA to generate a 3⬘-PO4/5-OH nick that can be repaired by the enzymes DNA 3⬘-phosphatase, DNA 5⬘-kinase, and DNA ligase. In vitro, topoisomerase IB can use water as a nucleophile to cleave the covalent intermediate and release a free 3⬘-PO4 DNA end, but the reaction is extremely slow and depends on nonphysiological alkaline pH conditions (Christiansen et al., 1994; Petersen and Shuman, 1997a). This reaction is not a reflection of the inherent chemical lability of the tyrosine phosphodiester; rather, hydrolysis is directly catalyzed by the topoisomerase active site (Krogh and Shuman, 2000b). Why then is topoisomerase not normally a more vigorous nuclease? An unexpected insight to this question emerges from an analysis of the effects of methylphosphonate modification of the scissile phosphodiester. We find that introduction of a methylphosphonate at the cleavage site transforms vaccinia topoisomerase into a potent site-specific endonuclease at physiological pH. The endonuclease reaction entails formation and rapid hydrolysis of a covalent enzyme-DNA methylphosphonate intermediate. Our results require a revised view of the catalytic mechanism of topoisomerase IB. The fact that transesterification occurs on the methylphosphonate DNAs attests to the lack of a stringent requirement for electrostatic interactions with the scissile phosphate in the ground state or for increased negative charge in the transition state. Moreover, the tremendous acceleration in the rate of hydrolysis of a methylphosphonate versus phosphodiester indicates that electrostatic repulsion of the water nucleophile is a major impediment to the latent nuclease activity of topoisomerase IB. Results Experimental Strategy to Probe the Influence of Charge and Polar Interactions of Topoisomerase at the Scissile Phosphodiester Vaccinia topoisomerase is a prototype of the type IB topoisomerase family, which includes eukaryotic nu-
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Figure 1. DNA Transesterification and Hydrolysis Reactions of Topoisomerase IB (A) The reaction pathway for transesterification at a DNA phosphodiester entails nucleophilic attack of tyrosine on the phosphorus to form a covalent DNA-(3⬘-PO4)-enzyme intermediate and a 5⬘-OH DNA strand. The reaction proceeds through a pentacoordinate phosphorane transition state. (B) The hypothetical reaction pathway for transesterification at a DNA methylphosphonate linkage. The methylphosphonate is uncharged in the ground state and has a nominal charge of ⫺1 in the proposed transition state. The reaction proceeds with inversion of stereochemical configuration at the chiral phosphorus center. (C) Attack by water on the covalent phosphodiester intermediate expels the tyrosine leaving group and yields a 3⬘-phosphate DNA strand. (D) Hydrolysis of the chiral covalent methylphosphonate intermediate generates an achiral 3⬘-methylphosphate DNA end.
clear and mitochondrial topoisomerases IB, other poxvirus topoisomerases, and poxvirus-like topoisomerases encoded by bacteria (Champoux, 2001; Zhang et al., 2001; Krogh and Shuman, 2002a). The vaccinia enzyme is distinguished by its compact size (314 amino acids) and its site specificity in DNA transesterification (Shuman, 1998). Vaccinia topoisomerase binds duplex DNA and nicks one strand at a pentapyrimidine target sequence, 5⬘-(T/C)CCTT↓ (Shuman and Prescott, 1990). The Tp↓ nucleotide (defined as the ⫹1 nucleotide) is linked to Tyr274 of the enzyme. Four conserved amino acid side chains found in all type IB topoisomerases (Arg130, Lys167, Arg223, and His265 in the vaccinia enzyme) are responsible for catalyzing the attack of the active site tyrosine nucleophile (Tyr274) on the scissile phosphodiester to form the covalent intermediate (Petersen and Shuman, 1997b; Wittschieben and Shuman, 1997; Cheng et al., 1997). Mutational, stereochemical, and structural data suggest
that Arg130 and His265 contact the nonbridging oxygens of the scissile phosphodiester and that these interactions serve to stabilize a proposed phosphorane transition state (Stivers et al., 2000; Krogh and Shuman, 2000b; Cheng et al., 1998; Redinbo et al., 1998) (Figure 1A). Lys167 and Arg130 comprise a proton relay that catalyzes the expulsion of the 5⬘-O of the leaving DNA strand (Krogh and Shuman, 2000a, 2002b). There is no requirement for, or participation of, a divalent cation in the transesterification reactions. The effects of DNA modifications on transesterification have provided insights to the topoisomerase reaction. For example, introduction of a 5⬘-phosphate/3⬘-OH nick in lieu of the scissile phosphodiester abolishes transesterification without affecting the noncovalent binding of topoisomerase to the nicked DNA (Cheng and Shuman, 1999). This result indicates that vaccinia topoisomerase is an obligate polynucleotidyl-3⬘-phosphotransferase and cannot transesterify to a 5⬘ phos-
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Figure 2. Sp Methylphosphonate Substitution at the Scissile Phosphodiester Converts Topoisomerase into an Endonuclease (A and B) The 18-mer/30-mer suicide substrate is illustrated at the bottom of the figure, and the cleavage site is indicated by an arrow. The scissile strand was 5⬘ 32P-labeled (indicated by an asterisk). Reaction mixtures contained 0.3 pmol DNA with an Sp methylphosphonate at the cleavage site and 2 pmol topoisomerase. Aliquots were withdrawn at the times specified and either (A) analyzed directly by SDS-PAGE or (B) digested with proteinase K and electrophoresed through a high-resolution TBE-urea sequencing gel. Autoradiographs of the gels are shown. The 5⬘ 32P-labeled reference oligonucleotides in the rightmost lanes of (B) were as follows: lane C, 5⬘-pCGTGTCGCCCTMePTATTCCCOH3⬘; lane M⬘, 5⬘-pCGTGTCGCMePCCTTp-3⬘; lane M, 5⬘-pCGTGTCGCCCTTp-3⬘.
phomonoester. Because a phosphodiester has a nominal net charge of ⫺1 in the ground state and ⫺2 in the proposed transition state (Figure 1A), whereas a 5⬘ phosphomonoester has a nominal charge of ⫺2 in the ground state (and would acquire a charge of ⫺3 in an associative transition state), it is possible that electrostatic repulsion of the attacking tyrosine nucleophile by the extra charge precludes a reaction with the 5⬘ monoester. Introduction of chiral phosphorothioates at the cleavage site elicits stereoselective reductions in the rate of transesterification (30-fold and 340-fold for the Sp and Rp diastereomers) without affecting the yield of the covalent intermediate (Stivers et al., 2000). The phosphorothioate modification imposes no change in net charge; however, the negative charge will localize largely on the sulfur, instead of distributing over the two nonbridging oxygens, as in a phosphodiester (Iyengar et al., 1984; Eckstein, 2000). The thio effects on cleavage rate are attributed to destabilization of the transition state, either via perturbation of the electrostatic environment or via steric hindrance imposed by the larger van der Waals radius of sulfur (1.85 A˚) compared to oxygen (1.44 A˚). Here, we use chiral methylphosphonate-substituted DNAs to probe the contributions of negative charge on the scissile phosphodiester and presumptive polar contacts of the enzyme with the nonbridging oxygens in the catalysis of the strand cleavage reaction. The methylphosphonate modification eliminates the negative charge on the phosphodiester in the ground state and would reduce the charge on the transition state
from ⫺2 to ⫺1 (Figure 1B). Also, the methyl group would not be able to engage in the hydrogen bonding interactions with arginine or histidine side chains that are invoked for the transition state. Sp Methylphosphonate Substitution at the Scissile Phosphodiester Converts Vaccinia Topoisomerase into a Nuclease We synthesized an 18-mer strand containing an Sp methylphosphonate at the CCCTTpA cleavage site. The methylphosphonate strand and an unmodified phosphodiester control strand were 5⬘ 32P-labeled and annealed to an unlabeled 30-mer complementary strand to form “suicide” substrates for vaccinia topoisomerase (Figure 2). Transesterification at the TpA linkage results in covalent attachment of a 5⬘ 32P-labeled 12-mer (5⬘pCGTGTCGCCCTTp-3⬘) to the enzyme via Tyr274. The unlabeled 6-mer 5⬘-OH leaving strand (5⬘-ATTCCC-3⬘) dissociates spontaneously from the protein-DNA complex and thereby drives the reaction toward the covalent state, so that the reaction can be treated kinetically as a unidirectional process (Stivers et al., 1994; Wittschieben and Shuman, 1997). Formation of the covalent adduct is detectable by SDS-PAGE analysis of the reaction products. The 32P-labeled DNA is transferred to the topoisomerase polypeptide to form an ⵑ40 kDa protein-DNA adduct that is well resolved from the DNA, which migrates near the dye front (Figure 2A). The reaction of topoisomerase with the unmodified phosphodiester substrate attained an endpoint of 93% covalent adduct within 20 s; from the extent of adduct formation at 5 s,
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we calculated a cleavage rate constant of 0.4 s⫺1. Note that the second order process of noncovalent binding of topoisomerase to the DNA target site is fast and saturating under the conditions used (e.g., the observed cleavage rate constant does not increase when the topoisomerase concentration is increased 2-fold), thus justifying the estimation of a pseudo-first order rate constant from the data. Analysis of product formation during a reaction of topoisomerase with the Sp methylphosphonate substrate revealed a transient accumulation of the covalent topoisomerase-DNA complex, which peaked at 3–5 min and declined steadily thereafter (Figure 2A). Concomitant with the disappearance of the topoisomerase-DNA adduct, there accumulated a novel 32P-labeled species that migrated at the solute front and was clearly separated from the input 18-mer oligonucleotide (Figure 2A). To identify the novel radiolabeled species, the reaction products were digested with proteinase K and then analyzed by electrophoresis through a 17% polyacrylamide/TBE/7 M urea gel that resolves DNAs on the basis of chain length. As shown in Figure 2B, the novel labeled product corresponds to a free 32P-labeled 12-mer DNA. The kinetics of accumulation of the 12-mer strand paralleled the appearance of the rapidly migrating product detected by SDS-PAGE. The analysis in Figure 2B also revealed the accumulation and disappearance of a cluster of radiolabeled products of proteolysis of the covalent intermediate, migrating between the 18-mer and 12-mer strands. This cluster (indicated by arrowheads in Figure 2B) consists of the 5⬘ 32P-labeled 12-mer strand linked to heterogeneous short peptides. Detection of the cluster in this gel system depended on proteinase K treatment, because the intact topoisomerase-DNA adduct does not enter the gel. The abundance of the labeled free 12-mer was not affected by omission of the proteolysis step (not shown). The kinetic profile of the formation of the covalent and free products (Figure 3A) is consistent with a precursorproduct relationship, whereby the topoisomerase first reacts with the Sp methylphosphonate substrate to form a DNA-(3⬘-methylphosphonate)-tyrosyl linkage, which is then hydrolyzed to liberate the free 12-mer DNA. The hydrolysis reaction is both efficient (95% of the input 18-mer strand being converted to the free 12-mer) and relatively rapid, being complete in 1 hr and attaining 50% of the endpoint in 10 min. The data in Figure 3A were fit by using the CKS kinetic simulation program (version 1.0; IBM) to a unidirectional two-step reaction mechanism with transesterification and hydrolysis rate constants of 0.0018 s⫺1 and 0.006 s⫺1, respectively (Figure 4). The apparent rate of transesterification of topoisomerase to the Sp methylphosphonate linkage was about 220-fold slower than the rate of covalent adduct formation at a phosphodiester (Figure 4). The reduced rate of topoisomerase transesterification to the Sp methylphosphonate DNA did not reflect interference with the noncovalent binding step, insofar as the observed rates of transesterification and hydrolysis did not increase when the concentration of topoisomerase was doubled (not shown).
Figure 3. Kinetic Profile of the Reaction of Topoisomerase with Methylphosphonate DNAs The abundance of the covalent methylphosphonate intermediate and the free 12-mer DNA (expressed as the percent of total 32P label in each species) is plotted as a function of time. (A) Sp methylphosphonate DNA. (B) Rp methylphosphonate DNA.
Different Effects of Sp and Rp Methylphosphonate Diastereomers We next analyzed the reaction of vaccinia topoisomerase with a 5⬘ 32P-labeled substrate containing an Rp methylphosphonate modification at the CCCTTpA cleavage site. Product analysis revealed a time-dependent accumulation of the rapidly migrating free DNA species at the solvent front, concomitant with the decay of the more slowly migrating substrate strand (Figure 5A). Trace levels of the covalent topoisomerase-DNA complex were seen from 2 to 120 min and declined at later times (see the top panel insert in Figure 5A). The covalent adduct to the Rp substrate comprised no more than ⵑ0.6% of the 32P label during this interval (compared to a peak value of 17% covalent adduct for the Sp substrate). Analysis of proteinase K-digested reaction products demonstrated the steady accumulation of a free 12-mer hydrolysis product (Figure 5B); in this exposure, the cluster of covalent 12-mer peptide adducts was not detectable.
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Figure 4. Rate Constants for Transesterification and Hydrolysis of Phosphodiester and Methylphosphonate DNAs by Wild-Type Vaccinia Topoisomerase and the H265A Mutant See the text for discussion. The structures of a standard DNA phosphodiester and the Sp and Rp methylphosphonate (MeP) diastereomers are illustrated. nd; not determined.
The kinetic profile of the hydrolysis of the Rp methylphosphonate is shown in Figure 3B. The extent of hydrolysis of the Rp substrate (ⵑ95%) was similar to that of the Sp diastereomer; however, the rate of Rp hydrolysis was obviously slower than the Sp reaction. Hydrolysis of the Rp DNA was complete in 8 hr and reached 50% of the endpoint in 2 hr (compared to 10 min for Sp DNA). The difference is attributable to a slower rate of transesterification by topoisomerase to the Rp methylphosphonate than to the Sp substrate. We did not detect a lag in the formation of the free 12-mer from the Rp DNA; this finding suggests a kinetic mechanism in which the rate constant for transesterification is at least 10fold slower than the rate of hydrolysis of the covalent intermediate. From a kinetic simulation, we estimated transesterification and hydrolysis rate constants of 0.00011 s⫺1 and 0.009 s⫺1, respectively (Figure 4). The rates of transesterification and hydrolysis on the Rp methylphosphonate DNA did not increase when the concentration of topoisomerase was doubled (not shown). Comparison of the rate constants for the Sp and Rp methylphosphonate substrates highlights a significant stereochemical effect on the transesterification step (Sp/Rp ⫽ 16), but not on the hydrolysis step. Because the transesterification reaction occurs with inversion of configuration at the phosphorus being attacked, the substrates for hydrolysis, which we presume to be the covalent DNA-(3⬘ methylphosphonate)-enzyme complex, will be of opposite chirality relative to the respective starting DNAs. Thus, hydrolysis of the Sp methylphosphonate DNA would entail attack of water on the stereochemically inverted diastereomer of the covalent intermediate, assuming that the covalent complex is an obligate intermediate along the hydrolysis reaction pathway. Hydrolysis Reaction Mechanism and Characterization of the Product If the methylphosphonate-dependent hydrolysis reactions occur via an obligate covalent intermediate, then
the active site tyrosine nucleophile (Tyr274) should be essential for the overall reactions with the Sp and Rp methylphosphonate substrates. On the other hand, if hydrolysis can occur via direct attack of water on the DNA methylphosphonate, then the tyrosine nucleophile might be dispensable. We gave serious consideration to the second pathway in light of studies of FLP recombinase showing that FLP mutants lacking the active site tyrosine, though inert in forming the covalent proteinDNA adduct, are nonetheless capable of hydrolyzing DNA in the presence of a strong nucleophile via direct attack on the scissile phosphodiester (Lee and Jayaram, 1993). Here, we found that the Y274A mutant of vaccinia topoisomerase was incapable of forming a free 12-mer strand when reacted with either the Sp or Rp methylphosphonate substrates during incubations of up to 24 hr (not shown), arguing that transesterification by Tyr274 at the methylphosphonate is a prerequisite for the observed hydrolysis reactions. In principle, the hydrolysis of the DNA-(3⬘-methylphosphotyrosyl)-enzyme linkage could yield either a DNA3⬘-methylphosphate strand plus the native tyrosyl enzyme or a DNA-3⬘-OH strand plus a phosphotyrosyl enzyme. The nature of the DNA 3⬘ terminus was characterized by electrophoresis of the 5⬘ 32P-labeled 12-mer hydrolysis product in parallel with a 5⬘ 32P-labeled 12mer of identical sequence that contained a 3⬘-phosphate end. The latter species (M in Figures 2B and 5B) was generated by peroxidolysis of the covalent adduct formed by topoisomerase on a standard phosphodiester substrate (Krogh and Shuman, 2000a). The methylphosphonate hydrolysis reaction products migrated just slightly slower than the 3⬘ phosphate-terminated 12-mer (Figures 2B and 5B). We surmised that this small mobility change reflected the difference in charge on the 3⬘ phosphate (net charge of ⫺2 at pH 8) versus a 3⬘ methylphosphate (net charge of ⫺1). This idea was supported by the finding that a 5⬘ 32P-labeled 12-mer of identical sequence containing a 3⬘-phosphate terminus and single internal methylphosphonate linkage (lane M in Figure 2B) mi-
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Figure 5. Reaction of Topoisomerase at an Rp Methylphosphonate Linkage (A and B) Reaction mixtures contained 0.3 pmol DNA with an Rp methylphosphonate at the cleavage site and 2 pmol topoisomerase. Aliquots were withdrawn at the times specified and either (A) analyzed directly by SDSPAGE or (B) digested with proteinase K and electrophoresed through a high-resolution DNA sequencing-type gel. Autoradiographs of the gels are shown. (A) includes a prolonged exposure of the SDS gel, revealing trace levels of the covalent intermediate. In (B), lane M contains the 5⬘-radiolabeled reference oligonucleotide pCGTGTCGCCCTTp.
grated identically to the methylphosphonate hydrolysis product (note that they have the same net charge on the 12-mer) and slightly slower than the unmodified 3⬘phosphate strand (lane M). The hydrolysis products of the Rp and Sp methylphosphonate DNAs migrated faster than a 5⬘ 32P-labeled 12-mer of identical sequence containing a 3⬘ hydroxyl terminus; this finding provides further evidence that the hydrolysis reaction yielded a 3⬘ methylphosphonate terminus (Figure 6, lane M). To confirm the assignments of the 3⬘ ends, we treated the Rp and Sp hydrolysis reaction products and the peroxidolysis reaction product (containing a 3⬘-phosphate terminus) with the enzyme T4 polynucleotide 3⬘ phosphatase. The peroxidolysis product was thereby converted quantitatively into a slower species that comigrated with the 3⬘-OH DNA standard. In contrast, neither of the methylphosphonate hydrolysis reaction products was affected by treatment with 3⬘ phosphatase, which attests to the fact that they have modified 3⬘-methylphosphate termini that are not susceptible to hydrolysis by the T4 enzyme.
Methylphosphonates Stimulate the Rate of Topoisomerase-Catalyzed Hydrolysis The rates of hydrolysis of the methylphosphonate-substituted covalent intermediates (0.006–0.009 s⫺1) are considerably faster than expected from previous estimates of the rate of topoisomerase-catalyzed hydrolysis
of the unmodified covalent phosphodiester intermediate under optimal alkaline pH conditions (Krogh and Shuman, 2000a). In order to directly evaluate the magnitude of the gain-of-function in hydrolysis elicited by the methylphosphonates, we determined the rate of hydrolysis of an unmodified suicide intermediate, comprising 95% of the total labeled DNA at 1 min after initiation of the reaction, by taking time points at 1-day intervals over a 7-day incubation a pH 7.5. The free 12-mer product accumulated progressively to an extent of 10% of the total labeled DNA after 7 days (data not shown). From the initial rate, we calculated a rate constant of 2.2 ⫻ 10⫺7 s⫺1 for hydrolysis of the DNA-3⬘-phosphotyrosyl linkage. Thus, replacement of a nonbridging oxygen on the covalent adduct by a methyl group accelerated the hydrolysis reaction by a factor of ⵑ30,000 (Figure 4). “Biochemical Epistasis” between His265 and the Sp Methyl Group His265 is an essential component of the topoisomerase active site that functions in transition state stabilization and not as a general acid-base catalyst (Krogh and Shuman, 2000a; Stivers et al., 2001). The fact that His265 could be replaced by either asparagine or glutamine suggested that this side chain contributes a neutral hydrogen bond to one of the nonbridging oxygens in the transition state (Petersen and Shuman, 1997b). The 200fold rate decrement in transesterification imposed by the Sp methyl group was quantitatively similar to the
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Figure 6. The Hydrolysis Reaction Product Is a DNA-3⬘-Methylphosphate The free 12-mer products of topoisomerase-catalyzed peroxidolysis of the covalent phosphodiester intermediate (control) and topo-catalyzed hydrolysis of the Rp and Sp methylphosphonate substrates were analyzed by denaturing PAGE either with (⫹) or without (⫺) prior treatment with T4 polynucleotide 3⬘-phosphatase. An autoradiograph of the gel is shown. The duration of the individual peroxidolysis or hydrolysis reactions was varied so as to attain near-quantitative conversion of the input 32P-labeled 18-mer strand (shown in the leftmost lane) to free 12-mer. The DNAs were recovered by phenol extraction and ethanol precipitation. Aliquots were incubated for 20 min at 37⬚C in phosphatase reaction mixtures (25 l) containing 100 mM imidazole (pH 6.0), 10 mM MgCl2, 10 mM -mercaptoethanol, 0.1 mg/ml BSA, and 10 ng purified recombinant T4 polynucleotide 3⬘-phosphatase. The digestion products were recovered by phenol extraction and ethanol precipitation. Lane M contained the 5⬘-radiolabeled reference oligonucleotide pCGTGTCGCCCTTOH.
effect of replacing catalytic residue His265 with alanine, i.e., the rate of single-turnover transesterification by the H265A mutant enzyme on the unmodified suicide substrate was 0.0018 s⫺1 (Figure 4). We therefore speculated that in the transition state, His265 forms a hydrogen bond to the oxygen corresponding to the Sp methyl. We tested this model by examining the reaction of the H265A mutant with the Rp and Sp methylphosphonatemodified DNAs. A prediction of the model is that if catalysis by His265 entails contact only with the pro Sp oxygen, then elimination of the His side chain should have little or no impact on the reaction of topoisomerase with the Sp methylphosphonate-substituted DNA. A corollary prediction is that the reaction of the Rp methylphosphonate substrate with topoisomerase should be slowed significantly by the loss of the stabilizing contact of His265 to the lone nonbridging oxygen. This reasoning echoes genetic epistasis analysis, whereby the effects of mutations in two genes in the same pathway are not exacerbated when combined, but mutations affecting parallel pathways are additive when combined. The extent and kinetic profile of the reaction of H265A with the Sp methylphosphonate DNA (Figure 7A) was nearly identical to that of wild-type topoisomerase on the same substrate (Figure 3A). The covalent intermedi-
Figure 7. Reaction of Topoisomerase Mutant H265A with Methylphosphonate DNAs (A and B) Reaction mixtures contained 0.3 pmol Sp or Rp methylphosphonate DNA and 2 pmol H265A topoisomerase. Aliquots were withdrawn at the times specified, digested with proteinase K, and then analyzed by TBE-urea PAGE. The product distributions are plotted as a function of time for the (A) Sp and (B) Rp substrates. No covalent intermediate was detected in the reaction of H265A with the Rp methylphosphonate substrate.
ate accumulated transiently at 2–5 min and decayed thereafter as free 12-mer was formed. From a kinetic simulation, we estimated transesterification and hydrolysis rate constants of 0.0024 s⫺1 and 0.0085 s⫺1, respectively (Figure 4). The H265A mutation had no deleterious effect on the rate of formation of the covalent complex on the Sp methylphosphonate DNA or its hydrolysis (Figure 4). The reaction profile of H265A on the Rp methylphosphonate DNA (Figure 7B) showed significant slowing of the composite transesterification/hydrolysis reaction compared to that of the wild-type enzyme on the same DNA (Figure 3B). The free 12-mer accumulated steadily over a 6-day incubation to an extent of 23% of the input-labeled scissile strand. We were unable to detect any accumulation of covalent intermediate at any of the time points analyzed (at a limit of detection of 0.2% of total labeled DNA). This lack of accumulation signified that the overall reaction rate was limited by the DNA transesterification step and that the rate of the hydrolysis step was much faster than the rate of transes-
Molecular Cell 206
terification. Thus, we fit the data to a single-exponential decay function and calculated a rate constant of 6 ⫻ 10⫺7 for formation of the 12-mer. This value can be taken as a reasonable approximation of the rate of transesterification of H265A at the Rp methylphosphonate linkage. Thus, the His265A mutation reduced kcl on the Rp DNA by a factor of 200 compared to that of wild-type topoisomerase (Figure 4). The magnitude of this decrement was nearly identical to that elicited by the H265A mutation on transesterification at an unmodified phosphodiester. These results provide evidence that the essential histidine of topoisomerase IB assists in catalysis of transesterification exclusively via contacts to the pro Sp oxygen of the scissile phosphodiester. They also illustrate the power of “biochemical epistasis” tests and chiral methylphosphonates to illuminate functionally relevant protein-DNA contacts at atomic resolution in the absence of an atomic structure. Discussion Chiral methylphosphonates have been used previously to test the effects of backbone charge neutralization on protein-nucleic acid binding interactions (Noble et al., 1984; Pritchard et al., 1994; Dertinger and Uhlenbeck, 2001), but they have not been applied widely to the analysis of phosphoryl transfer enzymes (Rosati et al., 2002). Topoisomerase IB is an excellent case study for the utility of the methylphosphonates to dissect an enzyme mechanism, insofar as the transesterification chemistry is independent of a metal cofactor and catalysis is presumed to entail stabilizing contacts between amino acids of the enzyme and the nonbridging phosphate oxygens of the proposed transition state. Here, we have shown that chiral methylphosphonate substitutions at the cleavage site elicit two highly instructive effects on vaccinia virus topoisomerase IB. First, the Sp and Rp methylphosphonate diastereomers slow the rate of DNA transesterification by factors of 220 and 3600, respectively. Second, they unmask a latent and highly potent hydrolytic activity, effectively converting the topoisomerase into a nuclease. The stereo-specific methylphosphonate interference effects on transesterification (Sp/Rp ⫽ 16) underscore the distinct contributions of electrostatic and/or hydrogen bonding interactions with the two different nonbridging phosphate oxygens. A simple interpretation is that each oxygen interacts with a different partner on the enzyme. The rate decrement associated with the Sp methyl group is quantitatively similar to the effect of replacing catalytic residue His265 with alanine (Petersen and Shuman, 1997b), and we have presented functional evidence that the histidine stabilizes the transition state by donating a hydrogen bond to the oxygen corresponding to the Sp methyl. This view is in keeping with the inferences of Stivers et al. (2000) concerning the contacts of His265 with the nonbridging oxygens, based on the effects of chiral phosphorothioate modifications at the cleavage site. (To avoid confusion in comparing the two studies, it must be kept in mind that the nomenclature for the Rp and Sp configurations of methylphosphonates is opposite to that of the chiral phosphorothioates, such that an Rp methylphosphonate is substituted at
the same oxygen as the Sp phosphorothioate and the Sp methylphosphonate is substituted at the same oxygen as an Rp phosphorothioate.) The fact that transesterification occurs as rapidly as it does on the Sp methylphosphonate DNA attests to the lack of a stringent requirement for ionic interactions between topoisomerase IB and the nonbridging phosphate oxygens in the ground state or for the obligate presence of two negative charges on the transition state. This contrasts with the recent report that DNA cleavage by the EcoRI restriction endonuclease is abolished absolutely by Rp or Sp methylphosphonate modification at the scissile phosphodiester (Rosati et al., 2002). One of the methylphosphonate diastereomers strongly affected the binding of EcoRI to its target site, but the other diastereomer did not. In the present study, the reduced rates of topoisomerase transesterification to the methylphosphonate-modified DNAs do not reflect interference with noncovalent binding, insofar as the reaction rates did not increase when the concentration of topoisomerase was doubled. The activation of the latent nuclease of vaccinia topoisomerase by methylphosphonates is the result of a 30,000- to 40,000-fold increase in the rate of self-catalyzed hydrolysis of the covalent methylphosphonate intermediate vis a vis the tyrosyl-DNA phosphodiester (Figures 1C and 1D). Whereas the phosphodiester intermediate has a half-life of 36 days at physiological pH, the methylphosphonate intermediate has a half-life of less than 2 min. This prodigious gain-of-function was independent of the stereochemical configuration of the methylphosphonate intermediate and is therefore likely to be attributable to altered electrostatics. Note that the accelerated hydrolysis reaction was not caused by an inherent chemical lability of the tyrosine-methylphosphonate-DNA linkage, insofar as the covalent intermediate formed by the wild-type or H256A topoisomerases at early times on the Sp methylphosphonate substrate (Figures 3A and 7A) was stable for at least 12 hr at room temperature after the topoisomerase was denatured with SDS. Further evidence that hydrolysis of the covalent methylphosphonate intermediate was catalyzed directly by the topoisomerase active site stems from our finding that a mutation of another catalytic amino acid (Arg223) elicited a 400-fold decrement in khydrol on the Rp methylphosphonate substrate (L.T. and S.S., unpublished data). A plausible explanation for the gain-of-function is that hydrolysis is normally strongly suppressed by electrostatic repulsion of the nucleophilic water and that neutralization of the negative charge by methyl substitution for either the pro Rp or pro Sp oxygens removes this barrier to attack by water on the covalent intermediate. It is truly remarkable that the rate of hydrolysis of the methylphosphonate intermediates by wild-type and H265A topoisomerase (0.006–0.009 s⫺1) was only two orders of magnitude slower than the normal religation step of the topoisomerase catalytic cycle (krel ⫽ 1 s⫺1), which entails attack on the covalent intermediate by a DNA 5⬘-OH that is optimally aligned at the active site by virtue of base pairing of the acceptor polynucleotide to the nonscissile strand. Note that the rate of hydrolysis of the methylphosphonate intermediates was as fast or
Latent Nuclease Activity of Topoisomerase IB 207
faster than DNA religation across a 1 nucleotide gap, where krel ⫽ 0.005 s⫺1 (Sekiguchi et al., 1997). Previous studies showed that the H265A mutation slowed the rate of self-catalyzed hydrolysis of the covalent phosphodiester intermediate at alkaline pH by nearly two orders of magnitude (Krogh and Shuman, 2000a). Given that the observed khydrol for the wild-type topoisomerase-DNA phosphodiester intermediate at pH 7.5 was 2.2 ⫻ 10⫺7 s⫺1, a conservative estimate is that the rate of hydrolysis by the H265A mutant under the present reaction conditions would be ⬍10⫺8 s⫺1 (Figure 4). Comparison to the observed khydrol of the Sp-modified 18-mer substrate by the H265A mutant (0.0085 s⫺1) highlights a gain of hydrolytic function for the mutant enzyme of about six orders of magnitude. In summary, methylphosphonates expose the Achilles’ heel of topoisomerase IB as a guardian of genomic integrity. The topoisomerase is capable of vigorous hydrolytic activity, but reaction of the covalent intermediate with water is suppressed by virtue of electrostatic repulsion at the active site. Simple neutralization of the charge on the scissile phosphate of the covalent intermediate elicits a quantitative gain-of-function that, to our knowledge, is unprecedented in an enzymatic phosphoryl transfer reaction. Our results focus attention on repulsion of the attacking nucleophile as a potentially limiting factor in other enzymatic reactions at phosphates (especially metal-independent reactions), and they underscore the power of chiral methylphosphonates to address this question. Experimental Procedures DNA Substrates Oligonucleotides containing single chiral methylphosphonates were prepared by incorporating isomerically pure Sp and Rp dinucleotide methylphosphonate 5⬘-TpA cassettes during standard phosphoramidite-based oligonucleotide synthesis protocols. Synthesis and purification of the TpA methylphosphonate cassettes were performed essentially as described (Reynolds et al., 1996). The methylphosphonate dinucleotides were ⬎95% diastereochemically pure, as determined by analytical HPLC with peak integration and 31P NMR spectroscopic analysis. Detailed methods for chemical synthesis will be reported separately. The CCCTT-containing scissile strands were 5⬘ 32P-labeled by enzymatic phosphorylation in the presence of [␥-32P] ATP and T4 polynucleotide kinase. The 18-mer labeled oligonucleotides were gel purified and annealed to an unlabeled 30-mer oligonucleotide (containing a 5⬘-PO4 terminus introduced during chemical synthesis) at a 1:4 molar ratio of 18mer:30-mer. Vaccinia Topoisomerase Recombinant vaccinia topoisomerase was produced in E. coli BL21 by infection with bacteriophage CE6 and then purified to apparent homogeneity from the soluble bacterial lysate by phosphocellulose and Source S-15 chromatography steps. Protein concentration was determined by using the dye binding method (Biorad) with bovine serum albumin as the standard. DNA Transesterification and Hydrolysis Reaction mixtures containing (per 20 l) 50 mM Tris-HCl (pH 7.5), 0.3 pmol 18-mer/30-mer DNA, and 75 ng vaccinia topoisomerase were incubated at 37⬚C. Aliquots (20 l) were withdrawn at the times specified and quenched immediately with SDS (1% final concentration). The products were analyzed by SDS-PAGE. Duplicate aliquots were quenched with SDS and were digested for 2 hr at 37⬚C with 10 g proteinase K. The mixtures were adjusted to 47% formamide, heat denatured, and electrophoresed through a 20% denaturing
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