Carbinolamine formation and dehydration in a DNA repair enzyme active site.

In order to suggest detailed mechanistic hypotheses for the formation and dehydration of a key carbinolamine intermediate in the T4 pyrimidine dimer glycosylase (T4PDG) reaction, we have investigated these reactions using steered molecular dynamics with a coupled quantum mechanics-molecular mechanics potential (QM/MM). We carried out simulations of DNA abasic site carbinolamine formation with and without a water molecule restrained to remain within the active site quantum region. We recovered potentials of mean force (PMF) from thirty replicate reaction trajectories using Jarzynski averaging. We demonstrated feasible pathways involving water, as well as those independent of water participation. The water-independent enzyme-catalyzed reaction had a bias-corrected Jarzynski-average barrier height of approximately (6.5 kcal mol(-1) (27.2 kJ mol(-1)) for the carbinolamine formation reaction and 44.5 kcal mol(-1) (186 kJ mol(-1)) for the reverse reaction at this level of representation. When the proton transfer was facilitated with an intrinsic quantum water, the barrier height was approximately 15 kcal mol(-1) (62.8 kJ mol(-1)) in the forward (formation) reaction and 19 kcal mol(-1) (79.5 kJ mol(-1)) for the reverse. In addition, two modes of unsteered (free dynamics) carbinolamine dehydration were observed: in one, the quantum water participated as an intermediate proton transfer species, and in the other, the active site protonated glutamate hydrogen was directly transferred to the carbinolamine oxygen. Water-independent unforced proton transfer from the protonated active site glutamate carboxyl to the unprotonated N-terminal amine was also observed. In summary, complex proton transfer events, some involving water intermediates, were studied in QM/MM simulations of T4PDG bound to a DNA abasic site. Imine carbinolamine formation was characterized using steered QM/MM molecular dynamics. Dehydration of the carbinolamine intermediate to form the final imine product was observed in free, unsteered, QM/MM dynamics simulations, as was unforced acid-base transfer between the active site carboxylate and the N-terminal amine.

Structure of T4 pyrimidine dimer glycosylase in a reduced imine covalent complex with abasic site-containing DNA.

The base excision repair (BER) pathway for ultraviolet light (UV)-induced cyclobutane pyrimidine dimers is initiated by DNA glycosylases that also possess abasic (AP) site lyase activity. The prototypical enzyme known to catalyze these reactions is the T4 pyrimidine dimer glycosylase (T4-Pdg). The fundamental chemical reactions and the critical amino acids that lead to both glycosyl and phosphodiester bond scission are known. Catalysis proceeds via a protonated imine covalent intermediate between the alpha-amino group of the N-terminal threonine residue and the C1' of the deoxyribose sugar of the 5' pyrimidine at the dimer site. This covalent complex can be trapped as an irreversible, reduced cross-linked DNA-protein complex by incubation with a strong reducing agent. This active site trapping reaction is equally efficient on DNA substrates containing pyrimidine dimers or AP sites. Herein, we report the co-crystal structure of T4-Pdg as a reduced covalent complex with an AP site-containing duplex oligodeoxynucleotide. This high-resolution structure reveals essential precatalytic and catalytic features, including flipping of the nucleotide opposite the AP site, a sharp kink (approximately 66 degrees ) in the DNA at the dimer site and the covalent bond linking the enzyme to the DNA. Superposition of this structure with a previously published co-crystal structure of a catalytically incompetent mutant of T4-Pdg with cyclobutane dimer-containing DNA reveals new insights into the structural requirements and the mechanisms involved in DNA bending, nucleotide flipping and catalytic reaction.

Modulation of the turnover of formamidopyrimidine DNA glycosylase.

In recent years, significant progress has been made in determining the catalytic mechanisms by which base excision repair (BER) DNA glycosylases and glycosylase-abasic site (AP) lyases cleave the glycosyl bond. While these investigations have identified active site residues and active site architectures, few investigations have analyzed postincision turnover events. Previously, we identified a critical residue (His16) in the T4-pyrimidine dimer glycosylase (T4-Pdg) that, when mutated, interferes with enzyme turnover [Meador et al. (2004) J. Biol. Chem. 279, 3348-3353]. To test whether comparable residues and mechanisms might be operative for other BER glycosylase:AP-lyases, molecular modeling studies were conducted comparing the active site regions of T4-Pdg and the Escherichia coli formamidopyrimidine DNA glycosylase (Fpg). These analyses revealed that His71 in Fpg might perform a similar function to His16 in T4-Pdg. Site-directed mutagenesis of the Fpg gene and analyses of the reaction mechanism of the mutant enzyme revealed that the H71A enzyme retained activity on a DNA substrate containing an 8-oxo-7,8-dihydroguanine (8-oxoG) opposite cytosine and DNA containing an AP site. The H71A Fpg mutant was severely compromised in enzyme turnover on the 8-oxoG-C substrate but had turnover rates comparable to that of wild-type Fpg on AP-containing DNA. The similar mutant phenotypes for these two enzymes, despite a complete lack of structural or sequence homology between them, suggest a common mechanism for the rate-limiting step catalyzed by BER glycosylase:AP-lyases.

Reaction intermediates in the catalytic mechanism of Escherichia coli MutY DNA glycosylase.

The Escherichia coli adenine DNA glycosylase, MutY, plays an important role in the maintenance of genomic stability by catalyzing the removal of adenine opposite 8-oxo-7,8-dihydroguanine or guanine in duplex DNA. Although the x-ray crystal structure of the catalytic domain of MutY revealed a mechanism for catalysis of the glycosyl bond, it appeared that several opportunistically positioned lysine side chains could participate in a secondary beta-elimination reaction. In this investigation, it is established via site-directed mutagenesis and the determination of a 1.35-A structure of MutY in complex with adenine that the abasic site (apurinic/apyrimidinic) lyase activity is alternatively regulated by two lysines, Lys142 and Lys20. Analyses of the crystallographic structure also suggest a role for Glu161 in the apurinic/apyrimidinic lyase chemistry. The beta-elimination reaction is structurally and chemically uncoupled from the initial glycosyl bond scission, indicating that this reaction occurs as a consequence of active site plasticity and slow dissociation of the product complex. MutY with either the K142A or K20A mutation still catalyzes beta and beta-delta elimination reactions, and both mutants can be trapped as covalent enzyme-DNA intermediates by chemical reduction. The trapping was observed to occur both pre- and post-phosphodiester bond scission, establishing that both of these intermediates have significant half-lives. Thus, the final spectrum of DNA products generated reflects the outcome of a delicate balance of closely related equilibrium constants.

Role of His-16 in turnover of T4 pyrimidine dimer glycosylase.

Previously, the histidine residue at position 16 in the mature T4 pyrimidine dimer glycosylase (T4-PDG) protein has been suggested to be involved in general (non-target) DNA binding. This interpretation is likely correct, but, in and of itself, cannot account for the most dramatic phenotype of mutants at this position: their inability to restore ultraviolet light resistance to a DNA repair-deficient Escherichia coli strain. Accordingly, this residue has been mutated to serine, glutamic, aspartic acid, lysine, cysteine, and alanine. The mutant proteins were expressed, purified, and their abilities to carry out several functions of T4-PDG were assessed. The mutant proteins were able to perform most functions tested in vitro, albeit at reduced rates compared with the wild type protein. The most likely explanation for the biochemical phenotypes of the mutants is that the histidine residue is required for rapid turnover of the enzyme. This role is interpreted and discussed in the context of a reaction mechanism able to account for the complete spectrum of products generated by T4-PDG during a single turnover cycle.

Evidence for multiple imino intermediates and identification of reactive nucleophiles in peptide-catalyzed beta-elimination at abasic sites.

Prior investigations have demonstrated that peptides containing a single aromatic residue flanked by basic ones, such as Lys-Trp-Lys, can incise the phosphodiester backbone of duplex DNA at an AP site via beta-elimination. An amine serves as the reactive nucleophile to attack C1' on the ring-open deoxyribose sugar to form a transient peptide-DNA imino (Schiff base) intermediate, which may be isolated as a stable covalent species under reducing conditions. In the current study, we use this methodology to demonstrate that peptide-catalyzed beta-elimination proceeds via the formation of two Schiff base intermediates, one of which was covalently trapped prior to strand incision and the other following strand incision. N-Terminal acetylation of reactive peptides significantly inhibited formation of a trapped Schiff base complex; thus, we demonstrate for the first time that the preferred reactive nucleophile for peptides catalyzing strand incision is the N-terminal alpha-amino group, not an epsilon-amino group located on a lysine residue as previously postulated. Trapping reactions in which the central tryptophan residue was changed to alanine did not have a significant impact on the efficiency of Schiff base formation, indicating that the presence of an aromatic residue is dispensable for the step prior to peptide-catalyzed beta-elimination. Interestingly, the methodology presented here affords a convenient means for covalently attaching an array of peptides onto AP site-containing DNA in a site-specific fashion. We suggest that the generation of such DNA-peptide cross-links may provide utility in studying the repair of biologically significant DNA-protein cross-link damage as DNA-peptide complexes may mimic intermediate structures along a repair pathway for DNA-protein cross-links.

Mechanistic comparisons among base excision repair glycosylases.

The mechanisms by which various DNA glycosylases initiate the base excision repair pathways are discussed. Fundamental distinctions are made between "simple glycosylases," that do not form DNA single-strand breaks, and "glycosylases/abasic site lyases," that do form single-strand breaks. Several groupings of BER substrate sites are defined and some interactions between these groupings and glycosylase mechanisms discussed. Two characteristics are proposed to be common among all BER glycosylases: a nucleotide flipping step that serves to expose the scissile glycosyl bond to catalysis, and a glycosylase transition state characterized by substantial tetrahedral character at the base glycosyl atom.

Determination of active site residues in Escherichia coli endonuclease VIII.

Endonuclease VIII from Escherichia coli is a DNA glycosylase/lyase that removes oxidatively damaged bases. EndoVIII is a functional homologue of endonuclease III, but a sequence homologue of formamidopyrimidine-DNA glycosylase (Fpg). Using multiple sequence alignments, we have identified six target residues in endoVIII that may be involved in the enzyme's glycosylase and/or lyase functions: the N-terminal proline, and five acidic residues that are completely conserved in the endoVIII-Fpg proteins. To investigate the contribution of these residues, site-directed mutagenesis was used to create seven mutants: P2T, E3D, E3Q, E6Q, D129N, D160N, and E174Q. Each mutant was assayed both for lyase activity on abasic (AP) sites and for glycosylase/lyase activity on 5-hydroxyuracil, thymine glycol, and gamma-irradiated DNA with multiple lesions. The P2T mutant did not have lyase or glycosylase/lyase activity but could efficiently form Schiff base intermediates on AP sites. E6Q, D129N, and D160N behaved essentially as endoVIII in all assays. E3D, E3Q, and E174Q retained significant AP lyase activity but had severely diminished or abolished glycosylase/lyase activities on the DNA lesions tested. These studies provide detailed predictions concerning the active site of endoVIII.

Backbone dynamics of DNA containing 8-oxoguanine: importance for substrate recognition by base excision repair glycosylases.

Except for the functional groups sited within the major or minor grooves, the bases of B-DNA are quite protected from the external environment. Enzymes that modify the bases often "flip out" the target into an extrahelical position before the chemistry step is carried out. Examples of this mechanism are the base excision repair glycosylases and the restriction enzyme methylases. The question arises about the mechanism of substrate recognition for these enzymes and how closely it is linked to the base flipping step. Molecular dynamics simulations (AMBER, PME electrostatics) of fully solvated, cation neutralized, DNA sequences containing 8-oxoguanine (8OG) and of appropriate normal (control) DNAs have been carried out. The dynamics trajectories were analyzed to identify those properties of the DNA structure in the vicinity of the altered base, or its dynamics, that could contribute to molecular discrimination between substrate and non-substrate DNA sites. The results predict that the FPG enzyme should flip out the cytosine base paired with the scissile 8OG, not the target base itself.

The reaction mechanism of DNA glycosylase/AP lyases at abasic sites.

DNA glycosylase and glycosylase/abasic (AP) lyases are the enzymes responsible for initiating the base excision repair pathway by recognizing the damaged target base and catalyzing the breakage of the base-sugar glycosyl bond. The subset of glycosylases that have an associated AP lyase activity also catalyze DNA strand breakage at the resulting or preexisting AP site via a beta-elimination reaction, proceeding from an enzyme-DNA imino intermediate. Two distinct mechanisms have been proposed for the formation of this intermediate. These mechanisms essentially differ in the nature of the first bond broken and the timing of the opening of the deoxyribose ring. The data presented here demonstrate that the combined rate of sugar ring opening and reduction of the sugar is significantly slower than the rate of formation of a T4-pyrimidine dimer glycosylase (T4-pdg)-DNA intermediate. Using a methyl-deoxyribofuranose AP-site analogue that is incapable of undergoing sugar ring opening, it was demonstrated that the T4-pdg reaction can initiate at the ring-closed form, albeit at a drastically reduced rate. T4-pdg preferentially cleaved the beta-anomer of the methyl-deoxyribofuranose AP site analogue. This is consistent with a mechanism in which the methoxy group is backside-displaced by the amino group from the alpha-face of the deoxyribofuranose ring. In addition, studies examining rates of sugar-aldehyde reduction and the sodium borohydride concentration dependence of the rate of formation of the covalent imine intermediate suggest that the reduction of the intermediate is rate-limiting in the reaction.

Initiation of base excision repair: glycosylase mechanisms and structures.

The base excision repair pathway is an organism's primary defense against mutations induced by oxidative, alkylating, and other DNA-damaging agents. This pathway is initiated by DNA glycosylases that excise the damaged base by cleavage of the glycosidic bond between the base and the DNA sugar-phosphate backbone. A subset of glycosylases has an associated apurinic/apyrimidinic (AP) lyase activity that further processes the AP site to generate cleavage of the DNA phosphate backbone. Chemical mechanisms that are supported by biochemical and structural data have been proposed for several glycosylases and glycosylase/AP lyases. This review focuses on the chemical mechanisms of catalysis in the context of recent structural information, with emphasis on the catalytic residues and the active site conformations of several cocrystal structures of glycosylases with their substrate DNAs. Common structural motifs for DNA binding and damage specificity as well as conservation of acidic residues and amino groups for catalysis are discussed.

Characterization of a novel cis-syn and trans-syn-II pyrimidine dimer glycosylase/AP lyase from a eukaryotic algal virus, Paramecium bursaria chlorella virus-1.

Endonuclease V from bacteriophage T4, is a cis-syn pyrimidine dimer-specific glycosylase. Recently, the first sequence homolog of T4 endonuclease V was identified from chlorella virus Paramecium bursaria chlorella virus-1 (PBCV-1). Here we present the biochemical characterization of the chlorella virus pyrimidine dimer glycosylase, cv-PDG. Interestingly, cv-PDG is specific not only for the cis-syn cyclobutane pyrimidine dimer, but also for the trans-syn-II isomer. This is the first trans-syn-II-specific glycosylase identified to date. Kinetic analysis demonstrates that DNAs containing both types of pyrimidine dimers are cleaved by the enzyme with similar catalytic efficiencies. Cleavage analysis and covalent trapping experiments demonstrate that the enzyme mechanism is consistent with the model proposed for glycosylase/AP lyase enzymes in which the glycosylase action is mediated via an imino intermediate between the C1' of the sugar and an amino group in the enzyme, followed by a beta-elimination reaction resulting in cleavage of the phosphodiester bond. cv-PDG exhibits processive cleavage kinetics which are diminished at salt concentrations greater than those determined for T4 endonuclease V, indicating a possibly stronger electrostatic attraction between enzyme and DNA. The identification of this new enzyme with broader pyrimidine dimer specificity raises the intriguing possibility that there may be other T4 endonuclease V-like enzymes with specificity toward other DNA photoproducts.

The role of base flipping in damage recognition and catalysis by T4 endonuclease V.

The process of moving a DNA base extrahelical (base flipping) has been shown in the co-crystal structure of a UV-induced pyrimidine dimer-specific glycosylase, T4 endonuclease V, with its substrate DNA. Compared with other enzymes known to use base flipping, endonuclease V is unique in that it moves the base opposite the target site extrahelical, rather than moving the target base itself. Utilizing substrate analogs and catalytically inactive mutants of T4 endonuclease V, this study investigates the discrete steps involved in damage recognition by this DNA repair enzyme. Specifically, fluorescence spectroscopy analysis shows that fluorescence changes attributable to base flipping are specific for only the base directly opposite either abasic site analogs or the 5'-thymine of a pyrimidine dimer, and no changes are detected if the 2-aminopurine is moved opposite the 3'-thymine of the pyrimidine dimer. Interestingly, base flipping is not detectable with every specific binding event suggesting that damage recognition can be achieved without base flipping. Thus, base flipping does not add to the stability of the specific enzyme-DNA complex but rather induces a conformational change to facilitate catalysis at the appropriate target site. When used in conjunction with structural information, these types of analyses can yield detailed mechanistic models and critical amino acid residues for extrahelical base movement as a mode of damage recognition.

Role of specific amino acid residues in T4 endonuclease V that alter nontarget DNA binding.

Endonuclease V is a pyrimidine dimer-specific DNA glycosylase-apurinic (AP)1 lyase which, in vivo or at low salt concentrations in vitro, binds nontarget DNA through electrostatic interactions and remains associated with that DNA until all dimers have been recognized and incised. On the basis of the analyses of previous mutants that effect this processive nicking activity, and the recently published cocrystal structure of a catalytically deficient endonuclease V with pyrimidine dimer-containing DNA [Vassylyev, D. G., et al. (1995) Cell 83, 773-782], four site-directed mutations were created, the mutant enzymes expressed in repair-deficient Escherichia coli, and the enzymes purified to homogeneity. Steady-state kinetic analyses revealed that one of the mutants, Q15R, maintained an efficiency (k(cat)/Km) near that of the wild-type enzyme, while R117N and K86N had a 5-10-fold reduction in efficiency and K121N was reduced almost 100-fold. In addition, K121N and K86N exhibited a 3-5-fold increase in Km, respectively. All the mutants experienced mild to severe reduction in catalytic activity (k(cat)), with K121N being the most severely affected (35-fold reduction). Two of the mutants, K86N and K121N, showed dramatic effects in their ability to scan nontarget DNA and processively incise at pyrimidine dimers in UV-irradiated DNA. These enzymes (K86N and K121N) appeared to utilize a distributive, three-dimensional search mechanism even at low salt concentrations. Q15R and R117N displayed somewhat diminished processive nicking activities relative to that of the wild-type enzyme. These results, combined with previous analyses of other mutant enzymes and the cocrystal structure, provide a detailed architecture of endonuclease V-nontarget DNA interactions.

Purification and cloning of Micrococcus luteus ultraviolet endonuclease, an N-glycosylase/abasic lyase that proceeds via an imino enzyme-DNA intermediate.

Although Micrococcus luteus UV endonuclease has been reported to be an 18-kDa enzyme with possible homology to the 16-kDa endonuclease V from bacteriophage T4 (Gordon, L. K., and Haseltine, W. A. (1980) J. Biol. Chem. 255, 12047-12050; Grafstrom, R. H., Park, L., and Grossman, L. (1982) J. Biol. Chem. 257, 13465-13474), this study describes three independent purification schemes in which M. luteus UV damage-specific or pyrimidine dimer-specific nicking activity was associated with two proteins of apparent molecular masses of 31 and 32 kDa. An 18-kDa contaminant copurified with the doublet through many of the chromatographic steps, but it was determined to be a homolog of Escherichia coli ribosomal protein L6. Edman degradation analyses of the active proteins yielded identical NH2-terminal amino acid sequences. The corresponding gene (pdg, pyrimidine dimer glycosylase) was cloned. The protein bears strong sequence similarities to the E. coli repair proteins endonuclease III and MutY. Nonetheless, traditionally purified M. luteus protein acted exclusively on cis-syn thymine dimers; it was unable to cleave site-specific oligonucleotide substrates containing a trans-syn -I, (6-4), or Dewar thymine dimer, a 5,6-dihydrouracil lesion, or an A:G or A:C mismatch. The UV endonuclease incised cis-syn dimer-containing DNA in a dose-dependent manner and exhibited linear kinetics within that dose range. Enzyme activity was inhibited by the presence of NaCN or NaBH4 with NaBH4 additionally being able to trap a covalent enzyme-substrate product. These last findings confirm that the catalytic mechanism of M. luteus UV endonuclease, like those of other glycosylase/AP lyases, involves an imino intermediate.

Studies on the catalytic mechanism of five DNA glycosylases. Probing for enzyme-DNA imino intermediates.

DNA glycosylases catalyze scission of the N-glycosylic bond linking a damaged base to the DNA sugar phosphate backbone. Some of these enzymes carry out a concomitant abasic (apyrimidinic/apurinic(AP)) lyase reaction at a rate approximately equal to that of the glycosylase step. As a generalization of the mechanism described for T4 endonuclease V, a repair glycosylase/AP lyase that is specific for ultraviolet light-induced cis-syn pyrimidine dimers, a hypothesis concerning the mechanism of these repair glycosylases has been proposed. This hypothesis describes the initial action of all DNA glycosylases as a nucleophilic attack at the sugar C-1' of the damaged base nucleoside, resulting in scission of the N-glycosylic bond. It is proposed that the enzymes that are only glycosylases differ in the chemical nature of the attacking nucleophile from the glycosylase/AP lyases. Those DNA glycosylases, which carry out the AP lyase reaction at a rate approximately equal to the glycosylase step, are proposed to use an amino group as the nucleophile, resulting in an imino enzyme-DNA intermediate. The simple glycosylases, lacking the concomitant AP lyase activity, are propose to use some nucleophile from the medium, e.g. an activated water molecule. This paper reports experimental tests of this hypothesis using five representative enzymes, and these data are consistent with this hypothesis.

Involvement of glutamic acid 23 in the catalytic mechanism of T4 endonuclease V.

Bacteriophage T4 endonuclease V has both pyrimidine dimer-specific DNA glycosylase and abasic (AP) lyase activities, which are sequential yet biochemically separable functions. Previous studies using chemical modification and site-directed mutagenesis techniques have shown that the catalytic activities are mediated through the alpha-amino group of the enzyme forming a covalent (imino) intermediate. However, in addition to the amino-terminal active site residue, examination of the x-ray crystal structure of endonuclease V reveals the presence of Glu-23 near the active site, and this residue has been strongly implicated in the reaction chemistry. In order to understand the role of Glu-23 in the reaction mechanism, four different mutations (E23Q, E23C, E23H, E23D) were constructed, and the mutant proteins were evaluated for DNA glycosylase and AP lyase activities using defined substrates and specific in vitro and in vivo assays. Replacement of Glu-23 with Gln, Cys, or His completely abolished DNA glycosylase and AP lyase activities, while replacement with Asp retained negligible amounts of glycosylase activity, but retained near wild type levels of AP lyase activity. Gel shift assays revealed that all four mutant proteins can recognize and bind to thymine dimers. The results indicate that Glu-23 is the candidate for stabilizing the charge of the imino intermediate that is likely to require an acidic group in the active site of the enzyme.

Evidence for an imino intermediate in the T4 endonuclease V reaction.

Reductive methylation and site-directed mutagenesis experiments have implicated the N-terminal alpha-amino group of T4 endonuclease V in the glycosylase and abasic lyase activities of the enzyme. NMR studies have confirmed the involvement of the N-terminal alpha-amino group in the inhibition of enzyme activity by reductive methylation. A mechanism accounting for these results predicts that a (imino) covalent enzyme-substrate intermediate is formed between the protein N-terminal alpha-amino group and C1' of the 5'-deoxyribose of the pyrimidine dimer substrate subsequent to (or concomitantly with) the glycosylase step. Experiments to verify the existence of this intermediate indicated that enzyme inhibition by cyanide was substrate-dependent, a result classically interpreted to imply an imino reaction intermediate. In addition, sodium borohydride reduction of the intermediate formed a stable dead-end enzyme-substrate product. This product was formed whether ultraviolet light-irradiated high molecular weight DNA or duplex oligonucleotides containing a defined thymine-thymine cyclobutane dimer were used as substrate. The duplex oligonucleotide substrates demonstrated a well-defined gel shift. This will facilitate high-resolution footprinting of the enzyme on the DNA substrate.