Recognition of the nonpolar base 4-methylindole in DNA by the DNA repair adenine glycosylase MutY

[formula: see text] The DNA repair adenine glycosylase MutY efficiently recognizes 7-deaza-2'-deoxyadenosine (Z) and its nonpolar isostere 4-methylindole beta-deoxynucleoside (M) opposite 7,8-dihydro-8-oxo-2'-deoxyguanosine (OG) and G in DNA. Both wild-type and truncated MutY exhibit a 10- to 20-fold higher affinity for a duplex containing OG:M than OG:Z. More efficient recognition of M over Z by MutY may be to due the lack of hydrogen bonding with the OG that facilitates nucleotide flipping during the substrate recognition process.     

Converting the sacrificial DNA repair protein N-ada into a catalytic methyl phosphotriester repair enzyme

Mutation of the active-site residue Cys38 of N-Ada converts it from a sacrificial DNA repair protein to an enzyme that uses methanethiol as an external sacrificial reagent to repair DNA methyl phosphotriesters catalytically.     

Characterization of the DNA repair spore photoproduct lyase enzyme from Clostridium acetobutylicum: A radical-SAM enzyme

Spore photoproduct lyase (SPL) is a “Radical-SAM” repair enzyme which catalyzes the cleavage of spore photoproduct (SP, 5-thyminyl-5,6-dihydrothymine), a specific lesion found in bacterial spore DNA, to thymine monomers by a free-radical mechanism. The enzyme requires S-adenosyl-l-methionine (SAM) and a [4Fe–4S] cluster for activity. SPL from Bacillus subtilis has been difficult to isolate and characterize due to problems with the solubility and stability of the overexpressed protein in Escherichia coli and the lability of the [Fe–S] cluster, even if the protein was purified under strictly anaerobic conditions. In order to overcome these problems we searched for another SPL enzyme and we found that the recombinant SPL enzyme from Clostridium acetobutylicum, isolated either aerobically or anaerobically from overexpressing E. coli, behaves more stably than the B. subtilis one. We report here a complete spectroscopic and biochemical characterization of this enzyme. In particular we show for the first time that, using HYSCORE spectroscopy, SAM binds to the cluster as observed in the case of other members of the “Radical-SAM” enzyme family such as the activases of pyruvate formate lyase and ribonucleotide reductase.     

Transition-state analysis of S. pneumoniae 5'-methylthioadenosine nucleosidase

Kinetic isotope effects (KIEs) and computer modeling are used to approximate the transition state of S. pneumoniae 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN). Experimental KIEs were measured and corrected for a small forward commitment factor. Intrinsic KIEs were obtained for [1'-3H], [1'-14C], [2'-3H], [4'-3H], [5'-3H(2)], [9-15N] and [Me-3H(3)] MTAs. The intrinsic KIEs suggest an SN1 transition state with no covalent participation of the adenine or the water nucleophile. The transition state was modeled as a stable ribooxacarbenium ion intermediate and was constrained to fit the intrinsic KIEs. The isotope effects predicted a 3-endo conformation for the ribosyl oxacarbenium-ion corresponding to H1'-C1'-C2'-H2' dihedral angle of 70 degrees. Ab initio Hartree-Fock and DFT calculations were performed to study the effect of polarization of ribosyl hydroxyls, torsional angles, and the effect of base orientation on isotope effects. Calculations suggest that the 4'-3H KIE arises from hyperconjugation between the lonepair (n(p)) of O4' and the sigma* (C4'-H4') antibonding orbital owing to polarization of the 3'-hydroxyl by Glu174. A [methyl-3H(3)] KIE is due to hyperconjugation between np of sulfur and sigma* of methyl C-H bonds. The van der Waal contacts increase the 1'-3H KIE because of induced dipole-dipole interactions. The 1'-3H KIE is also influenced by the torsion angles of adjacent atoms and by polarization of the 2'-hydroxyl. Changing the virtual solvent (dielectric constant) does not influence the isotope effects. Unlike most N-ribosyltransferases, N7 of the leaving group adenine is not protonated at the transition state of S. pneumoniae MTAN. This feature differentiates the S. pneumoniae and E. coli transition states and explains the 10(3)-fold decrease in the catalytic efficiency of S. pneumoniae MTAN relative to that from E. coli.     

Transition-state analysis of Trypanosoma cruzi uridine phosphorylase-catalyzed arsenolysis of uridine

Uridine phosphorylase catalyzes the reversible phosphorolysis of uridine and 2'-deoxyuridine to generate uracil and (2-deoxy)ribose 1-phosphate, an important step in the pyrimidine salvage pathway. The coding sequence annotated as a putative nucleoside phosphorylase in the Trypanosoma cruzi genome was overexpressed in Escherichia coli , purified to homogeneity, and shown to be a homodimeric uridine phosphorylase, with similar specificity for uridine and 2'-deoxyuridine and undetectable activity toward thymidine and purine nucleosides. Competitive kinetic isotope effects (KIEs) were measured and corrected for a forward commitment factor using arsenate as the nucleophile. The intrinsic KIEs are: 1'-(14)C = 1.103, 1,3-(15)N(2) = 1.034, 3-(15)N = 1.004, 1-(15)N = 1.030, 1'-(3)H = 1.132, 2'-(2)H = 1.086, and 5'-(3)H(2) = 1.041 for this reaction. Density functional theory was employed to quantitatively interpret the KIEs in terms of transition-state structure and geometry. Matching of experimental KIEs to proposed transition-state structures suggests an almost synchronous, S(N)2-like transition-state model, in which the ribosyl moiety possesses significant bond order to both nucleophile and leaving groups. Natural bond orbital analysis allowed a comparison of the charge distribution pattern between the ground-state and the transition-state models.     

Characterization of nucleobase-amino acid stacking interactions utilized by a DNA repair enzyme

The present work characterizes the gas-phase stacking interactions between four aromatic amino acid residues (histidine, phenylalanine, tyrosine, and tryptophan) and adenine or 3-methyladenine due to the proposed utilization of these interactions by enzymes that repair DNA alkylation damage. The MP2 potential energy surfaces of the stacked dimers are considered as a function of four variables (vertical displacement, angle of rotation, horizontal displacement, and tilt angle) using a variety of basis sets. It is found that the maximum stacking interaction energy decreases with the amino acid according to TRP > TYR approximately HIS > PHE for both nucleobases. However, the magnitude of the stacking interaction significantly increases upon alkylation (by 50-115%). Comparison of the stacking energies calculated using our surface scans to those estimated from experimental crystal structures indicates that the stacking interactions within the active site of 3-methyladenine DNA glycosylase can account for 65-75% of the maximum possible stacking interaction between the relevant molecules. The decrease in stacking in the crystal structure arises due to significant differences in the relative orientations of the nucleobase and amino acid. Nevertheless, alkylation is found to significantly increase the stacking energy when the crystal structure geometries are considered. Our calculations provide computational support for suggestions that alkylation enhances the stacking interactions within the active site of DNA repair enzymes, and they give a measure of the magnitude of this enhancement. Our results suggest that alkylation likely plays a more important role in substrate identification and removal than the nature of the aromatic amino acid that interacts with the substrate via stacking interactions.     

Catalytic contributions of key residues in the adenine glycosylase MutY revealed by pH-dependent kinetics and cellular repair assays

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Highlights? Asp 138 must be deprotonated to stabilize the transition state for maximal activity ? Consequences of altering catalytic amino acids in MutY on cellular mismatch repair ? Correlation of MutY enzymatic parameters with ability to mediate cellular repair ? Importance of high affinity binding of MutY to the OG:A mismatch for high levels of repair     

Ultraviolet-B-induced inactivation of human OGG1, the repair enzyme for removal of 8-oxoguanine in DNA

The OGG1 proteins are DNA N-glycosylases-apurinic-apyrimidinic lyases that are responsible for the removal of 8-oxo-7,8-dihydroguanine (8-oxoG) base in DNA. The human enzyme (hOGG1) is a monomer of 345 amino acids containing 10 buried tryptophan (Trp) residues that are very sensitive to UVB irradiation. The photolysis quantum yield of these Trp residues is about 0.3 and 0.1 in argon- and air-saturated solutions, respectively. Matrix-assisted laser desorption-ionization-time-of-flight mass spectrometry shows that several cleavage sites are identical under aerobic and anaerobic photolysis of Trp residues; one of them includes the active site. Western blots and polyacrylamide gel electrophoresis indicate that fragments of high molecular size are also formed. In addition to common photochemical paths with argon-saturated solutions, specific reactions occur in air-saturated solutions of hOGG1. The photolysis rate is inhibited by more than 50% on binding of hOGG1 to a 34mer oligonucleotide containing a single 8-oxoG-C base pair. Binding to the oligonucleotide with 8-oxoG-C induced a 20% quenching of the hOGG1 fluorescence, suggesting interaction of nucleic acid bases with the Trp residue(s) responsible for the photolysis. Using 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine (Me-FapyG) and 8-oxoG as substrates, it is shown that protein photolysis induces photoinactivation of the DNA N-glycosylase activities. The excision of 8-oxoG is more affected than that of Me-FapyG at the same dose of UVB irradiation under both air and argon conditions. Besides the role of Trp residues, the possible involvement of Cys 253 in the photoinactivation process of hOGG1 is discussed.     

Preparation of single-stranded PCR products for electrospray ionization mass spectrometry using the DNA repair enzyme lambda exonuclease

Electrospray ionization mass spectrometry (ESI-MS) has been utilized to obtain accurate mass measurements of intact PCR products; however, single-stranded PCR products are necessary to detect sequence modifications such as base substitutions, additions or deletions. The locations of these modifications can subsequently be determined using additional stages of mass spectrometry. The recombinant enzyme lambda exonuclease selectively digests one strand of a DNA duplex from a 5' phosphorylated end leaving the complementary strand intact. Using this rapid enzymatic step, we were able to produce single-stranded PCR products by digestion of an intact PCR product derived from the Human Tyrosine Hydroxylase (HUMTHO1) gene, which contains a tetrameric repeating motif. The non-template directed 3' adenylation common when using Taq polymerase resulted in three distinct species (blunt-ended, mono-adenylated and di-adenylated), which added complexity to the spectrum of the double-stranded product. The data from the single-stranded products shows that one strand is preferentially adenylated over the other, which cannot be determined from the mass spectrum of the double-stranded PCR product alone. The ESI-FTICR (Fourier transform ion cyclotron resonance) mass spectra of the lambda exonuclease treated PCR products exhibited less than expected signal-to-noise (S/N) ratios. This is attributed to inaccurate concentration calculations due to remaining double-stranded PCR product amplified with unphosphorylated primers, and to matrix effects contributed by the lambda exonuclease reaction buffer. To further test this hypothesis, we investigated and determined the limit of detection to be 0.27 microM using standard curve statistics for single acquisitions of a synthetic 75-mer. The concentrations of the noncoding and coding strands produced by lambda exonuclease digestion were calculated to be 0.29 and 0.37 microM, respectively, taking into account the presence of double-stranded product. The products were electrosprayed from concentrations at the limit of detection requiring the averaging of 5-10 acquisitions to produce a sufficient S/N ratio, indicating that product concentration, base composition and matrix effects play a combined, significant role in detection of lambda exonuclease treated PCR products. Although additional work will be required to further exploit this strategy, lambda exonuclease clearly provides mass spectrometrists with a method to generate single-stranded PCR products.     

Transition-state structures for describing the enzyme-catalyzed mechanisms of rubisco

The carboxylation and oxygenation processes of a model substrate, 3,4-dihydroxy-2-pentanone, have been theoreticaly characterized as a set of steps, mimicking the corresponding reactions of D-ribulose-1,5-bisphosphate catalyzed by rubisco. A theoretical characterization is carried out of transition-state structures and possible molecular intermediates represented as saddle points of index 1 and minimum energy structures, respectively. The quantum chemical characterization, at the HF/3-21G calculation level, of these stationary points is used to rationalize and to discuss both catalyzed sequences. The reported set of these stationary points maps out most experimental aspects of the reaction pathways for the real system. Received: 24 March 1998 / Accepted: 3 September 1998 / Published online: 10 December 1998     

Transition-state structure of human 5'-methylthioadenosine phosphorylase

Kinetic isotope effects (KIEs) and computer modeling using density functional theory were used to approximate the transition state of human 5'-methylthioadenosine phosphorylase (MTAP). KIEs were measured on the arsenolysis of 5'-methylthioadenosine (MTA) catalyzed by MTAP and were corrected for the forward commitment to catalysis. Intrinsic KIEs were obtained for [1'-(3)H], [1'-(14)C], [2'-(3)H], [4'-(3)H], [5'-(3)H(2)], [9-(15)N], and [Me-(3)H(3)] MTAs. The primary intrinsic KIEs (1'-(14)C and 9-(15)N) suggest that MTAP has a dissociative S(N)1 transition state with its cationic center at the anomeric carbon and insignificant bond order to the leaving group. The 9-(15)N intrinsic KIE of 1.039 also establishes an anionic character for the adenine leaving group, whereas the alpha-primary 1'-(14)C KIE of 1.031 indicates significant nucleophilic participation at the transition state. Computational matching of the calculated EIEs to the intrinsic isotope effects places the oxygen nucleophile 2.0 Angstrom from the anomeric carbon. The 4'-(3)H KIE is sensitive to the polarization of the 3'-OH group. Calculations suggest that a 4'-(3)H KIE of 1.047 is consistent with ionization of the 3'-OH group, indicating formation of a zwitterion at the transition state. The transition state has cationic character at the anomeric carbon and is anionic at the 3'-OH oxygen, with an anionic leaving group. The isotope effects predicted a 3'-endo conformation for the ribosyl zwitterion, corresponding to a H1'-C1'-C2'-H2' torsional angle of 33 degrees. The [Me-(3)H(3)] and [5'-(3)H(2)] KIEs arise predominantly from the negative hyperconjugation of the lone pairs of sulfur with the sigma (C-H) antibonding orbitals. Human MTAP is characterized by a late S(N)1 transition state with significant participation of the phosphate nucleophile.     

Direct repair of the exocyclic DNA adduct 1,N6-ethenoadenine by the DNA repair AlkB proteins

The exocyclic DNA base adduct 1,N6-ethenoadenine (epsilonA) is directly repaired by the AlkB proteins in vitro.     

For MutY, it's all about the OG

MutY and its human ortholog, MUTYH, repair a specific form of DNA damage: adenine mis-paired with the oxidatively modified form of deoxyguanosine, 8-oxo-7,8-dihydro-2'-deoxyguanosine. In a recent issue of Chemistry & Biology, Brinkmeyer et?al. utilized mutant forms of MutY to reveal the critical residues in MutY that are required for this selectivity and specificity.     

Acetoaminophen-induced accumulation of 8-oxodeoxyguanosine through reduction of Ogg1 DNA repair enzyme in C6 glioma cells

Large doses of acetaminophen (APAP) could cause oxidative stress and tissue damage through production of reactive oxygen/nitrogen (ROS/RNS) species and quinone metabolites of APAP. Although ROS/RNS are known to modify DNA, the effect of APAP on DNA modifications has not been studied systematically. In this study, we investigate whether large doses of APAP can modify the nuclear DNA in C6 glioma cells used as a model system, because these cells contain cytochrome p450-related enzymes responsible for APAP metabolism and subsequent toxicity (Geng and Strobel, 1995). Our results revealed that APAP produced ROS and significantly elevated the 8-oxo- deoxyguanosine (8-oxodG) levels in the nucleus of C6 glioma cells in a time and concentration dependent manner. APAP significantly reduced the 8- oxodG incision activity in the nucleus by decreasing the activity and content of a DNA repair enzyme, Ogg1. These results indicate that APAP in large doses can increase the 8-oxodG level partly through significant reduction of Ogg1 DNA repair enzyme.     

Insights into the role of Val45 and Gln182 of Escherichia coli MutY in DNA substrate binding and specificity

Background  

Escherichia coli MutY (EcMutY) reduces mutagenesis by removing adenines paired with guanines or 7,8-dihydro-8-oxo-guanines (8-oxoG). V45 and Q182 of EcMutY are considered to be the key determinants of adenine specificity. Both residues are spatially close to each other in the active site and are conserved in MutY family proteins but not in Methanobacterium thermoautotrophicum Mig.MthI T/G mismatch DNA glycosylase (A50 and L187 at the corresponding respective positions).     

Chemistry and biology of DNA repair

Numerous agents of endogenous and exogenous origin damage DNA in our genome. There are several DNA-repair pathways that recognize lesions in DNA and remove them through a number of diverse reaction sequences. Defects in DNA-repair proteins are associated with several human hereditary syndromes, which show a marked predisposition to cancer. Although DNA repair is essential for a healthy cell, DNA-repair enzymes counteract the efficiency of a number of important antitumor agents that exert their cytotoxic effects by damaging DNA. DNA-repair enzymes are therefore also targets for drug design. DNA-repair processes differ greatly in their nature and complexity. Whereas some pathways only require a single enzyme to restore the original DNA sequence, others operate through the coordinated action of 30 or more proteins. Our understanding of the genetic, biochemical, and structural basis of DNA repair and related processes has increased dramatically over the past decade. This review summarizes the latest developments in this field.