1,N6-α-hydroxypropanoadenine, the acrolein adduct to adenine, is a substrate for AlkB dioxygenase.

1,N6-α-hydroxypropanoadenine (HPA) is an exocyclic DNA adduct of acrolein - an environmental pollutant and endocellular oxidative stress product. Escherichia coli AlkB dioxygenase belongs to the superfamily of α-ketoglutarate (αKG)- and iron-dependent dioxygenases which remove alkyl lesions from bases via an oxidative mechanism, thereby restoring native DNA structure. Here, we provide in vivo and in vitro evidence that HPA is mutagenic and is effectively repaired by AlkB dioxygenase. HPA generated in plasmid DNA caused A → C and A → T transversions and, less frequently, A → G transitions. The lesion was efficiently repaired by purified AlkB protein; the optimal pH, Fe(II), and αKG concentrations for this reaction were determined. In vitro kinetic data show that the protonated form of HPA is preferentially repaired by AlkB, albeit the reaction is stereoselective. Moreover, the number of reaction cycles carried out by an AlkB molecule remains limited. Molecular modeling of the T(HPA)T/AlkB complex demonstrated that the R stereoisomer in the equatorial conformation of the HPA hydroxyl group is strongly preferred, while the S stereoisomer seems to be susceptible to AlkB-directed oxidative hydroxylation only when HPA adopts the syn conformation around the glycosidic bond. In addition to the biochemical activity assays, substrate binding to the protein was monitored by differential scanning fluorimetry allowing identification of the active protein form, with cofactor and cosubstrate bound, and monitoring of substrate binding. In contrast FTO, a human AlkB homolog, failed to bind an ssDNA trimer carrying HPA.

[Synthesis, conformation, and spectroscopy of nucleoside analogues concerning their antiviral activity]. / Synteza, konformacja i spektroskopia analogów nukleozydów oraz ich aktywnosc antywirusowa.

Chemically modified analogues of nucleosides and nucleotides, have been thoroughly investigated since the discovery of DNA double helix by Watson and Crick in 1953 (Nature 171: 737). Chemical structures, first of all tautomerism, of the nucleic acid bases, as well as the conformations of the nucleic acids constituents, determine the secondary and tertiary structures of DNA and RNA polymers. Similarly, structural and dynamic parameters of nucleoside derivatives determine their biological activity in mutagenesis, neoplastic transformation, as well as antiviral or anticancer properties. In this review, a multidisciplinary approach of Prof. David Shugar's group is presented in the studies on nucleosides and nucleotides. It consists in chemical syntheses of suitable analogues, measurements of physicochemical and spectral parameters, conformational analysis by means of nuclear magnetic resonance (NMR) and X-ray diffraction, as well as characteristics of the nucleoside analogues as inhibitors of some selected, target enzymes, crucial in respect to antiviral activity of the analogues. These long-lasting studies follows upon the line of the main paradigm of molecular biophysics, i. e. structure-activity relationship.

AlkB dioxygenase preferentially repairs protonated substrates: specificity against exocyclic adducts and molecular mechanism of action.

Efficient repair by Escherichia coli AlkB dioxygenase of exocyclic DNA adducts 3,N(4)-ethenocytosine, 1,N(6)-ethenoadenine, 3,N(4)-α-hydroxyethanocytosine, and reported here for the first time 3,N(4)-α-hydroxypropanocytosine requires higher Fe(II) concentration than the reference 3-methylcytosine. The pH optimum for the repair follows the order of pK(a) values for protonation of the adduct, suggesting that positively charged substrates favorably interact with the negatively charged carboxylic group of Asp-135 side chain in the enzyme active center. This interaction is supported by molecular modeling, indicating that 1,N(6)-ethenoadenine and 3,N(4)-ethenocytosine are bound to AlkB more favorably in their protonated cationic forms. An analysis of the pattern of intermolecular interactions that stabilize the location of the ligand points to a role of Asp-135 in recognition of the adduct in its protonated form. Moreover, ab initio calculations also underline the role of substrate protonation in lowering the free energy barrier of the transition state of epoxidation of the etheno adducts studied. The observed time courses of repair of mixtures of stereoisomers of 3,N(4)-α-hydroxyethanocytosine or 3,N(4)-α-hydroxypropanocytosine are unequivocally two-exponential curves, indicating that the respective isomers are repaired by AlkB with different efficiencies. Molecular modeling of these adducts bound by AlkB allowed evaluation of the participation of their possible conformational states in the enzymatic reaction.

The role of AlkB protein in repair of 1,N6-ethenoadenine in Escherichia coli cells.

Etheno (ε) DNA adducts, including 1,N(6)-ethenoadenine (εA), are formed by various bifunctional agents of exogenous and endogenous origin. The AT→TA transversion, the most frequent mutation provoked by the presence of εA in DNA, is very common in critical codons of the TP53 and RAS genes in tumours induced by exposure to carcinogenic vinyl compounds. Here, using a method that allows examination of the mutagenic potency of a metabolite of vinyl chloride, chloroacetaldehyde (CAA), but eliminates its cytotoxicity, we studied the participation of alkA, alkB and mug gene products in the repair of εA in Escherichia coli cells. The test system used comprised the pIF105 plasmid bearing the lactose operon of CC105 origin, which allowed monitoring of Lac(+) revertants that arose by AT→TA substitutions due to the modification of adenine by CAA. The plasmid was CAA-modified in vitro and replicated in E.coli of various genetic backgrounds (wt, alkA, alkB, mug, alkAalkB, alkAmug and alkBmug). To modify the levels of the AlkA and AlkB proteins, mutagenesis was studied in E.coli cells induced or not in adaptive response to alkylating agents. Considering the levels of CAA-induced Lac(+) revertants in strains harbouring the CAA-modified pIF105 plasmid and induced or not in adaptive response, we conclude that the AlkB dioxygenase plays a major role in decreasing the level of AT→TA mutations, thus in the repair of εA in E.coli cells. The observed differences of mutation frequencies in the various mutant strains assayed indicate that Mug glycosylase is also engaged in the repair of εA, whereas the role the AlkA glycosylase in this repair is negligible.

Caenorhabditis elegans NDX-4 is a MutT-type enzyme that contributes to genomic stability.

MutT enzymes prevent DNA damage by hydrolysis of 8-oxodGTP, an oxidized substrate for DNA synthesis and antimutagenic, anticarcinogenic, and antineurodegenerative functions of MutT enzymes are well established. MutT has been found in almost all kingdoms of life, including many bacterial species, yeasts, plants and mammals. However, a Caenorhabditis elegans MutT homologue was not previously identified. Here, we demonstrate that NDX-4 exhibits both hallmarks of a MutT-type enzyme with an ability to hydrolyze 8-oxodGTP and suppress the Escherichia coli mutT mutator phenotype. Moreover, we show that NDX-4 contributes to genomic stability in vivo in C. elegans. Phenotypic analyses of an ndx-4 mutant reveal that loss of NDX-4 leads to upregulation of key stress responsive genes that likely compensate for the in vivo role of NDX-4 in protection against deleterious consequences of oxidative stress. This discovery will enable us to use this extremely robust genetic model for further research into the contribution of oxidative DNA damage to phenotypes associated with oxidative stress.

Chloroacetaldehyde-induced mutagenesis in Escherichia coli: the role of AlkB protein in repair of 3,N(4)-ethenocytosine and 3,N(4)-alpha-hydroxyethanocytosine.

Etheno (epsilon) adducts are formed in reaction of DNA bases with various environmental carcinogens and endogenously created products of lipid peroxidation. Chloroacetaldehyde (CAA), a metabolite of carcinogen vinyl chloride, is routinely used to generate epsilon-adducts. We studied the role of AlkB, along with AlkA and Mug proteins, all engaged in repair of epsilon-adducts, in CAA-induced mutagenesis. The test system used involved pIF102 and pIF104 plasmids bearing the lactose operon of CC102 or CC104 origin (Cupples and Miller (1989) [17]) which allowed to monitor Lac(+) revertants, the latter arose by GC-->AT or GC-->TA substitutions, respectively, as a result of modification of guanine and cytosine. The plasmids were CAA-damaged in vitro and replicated in Escherichia coli of various genetic backgrounds. To modify the levels of AlkA and AlkB proteins, mutagenesis was studied in E. coli cells induced or not in adaptive response. Formation of varepsilonC proceeds via a relatively stable intermediate, 3,N(4)-alpha-hydroxyethanocytosine (HEC), which allowed to compare repair of both adducts. The results indicate that all three genes, alkA, alkB and microg, are engaged in alleviation of CAA-induced mutagenesis. The frequency of mutation was higher in AlkA-, AlkB- and Mug-deficient strains in comparison to alkA(+), alkB(+), and microg(+) controls. Considering the levels of CAA-induced Lac(+) revertants in strains harboring the pIF plasmids and induced or not in adaptive response, we conclude that AlkB protein is engaged in the repair of epsilonC and HEC in vivo. Using the modified TTCTT 5-mers as substrates, we confirmed in vitro that AlkB protein repairs epsilonC and HEC although far less efficiently than the reference adduct 3-methylcytosine. The pH optimum for repair of HEC and epsilonC is significantly different from that for 3-methylcytosine. We propose that the protonated form of adduct interact in active site of AlkB protein.

Cockayne syndrome group B protein is engaged in processing of DNA adducts of lipid peroxidation product trans-4-hydroxy-2-nonenal.

Cockayne syndrome complementation group B (CSB) protein is engaged in transcription-coupled repair (TCR) of UV induced DNA damage and its deficiency leads to progressive multisystem degeneration and premature aging. Here, we show that human CSB-deficient cells are hypersensitive to physiological concentrations (1-10 microM) of a lipid peroxidation product, trans-4-hydroxy-2-nonenal (HNE), and in response to HNE they develop a higher level of sister chromatid exchanges (SCEs) in comparison to the wild-type cells. HNE-DNA adducts block in vitro transcription by T7 RNA polymerase, as well as by HeLa cell-free extracts. Treatment of wild-type cells with 1-20 microM HNE causes dephosphorylation of the CSB protein, which stimulates its ATPase activity necessary for TCR. However, high HNE concentrations (100-200 microM) inhibit in vitro CSB ATPase activity as well as the transcription machinery in HeLa cell-free extracts. Cell lines expressing CSB protein mutated in different ATPase domains exhibit different sensitivities to HNE. The motif II mutant, which binds ATP, but is defective in ATP hydrolysis was as sensitive to HNE as CSB-null cells. In contrast, motif V mutant cells were as sensitive to HNE as were the cells bearing wild-type protein, while motif VI mutant cells showed intermediate sensitivity to HNE. These mutants exhibit decreased ATP binding, but retain residual ATPase activity. Homology modeling suggested that amino acids mutated in motifs II and VI are localized closer to the ATP binding site than amino acids mutated in ATPase motif V. These results suggest that HNE-DNA adducts are extremely toxic endogenous DNA lesion, and that their processing involves CSB. When these lesions are not removed from the transcribed DNA strand due to CSB gene mutation or CSB protein inactivation by high, pathological HNE concentrations, they may contribute to accelerated aging.

Practical highly enantioselective synthesis of (R)- and (S)-(E)-4-hydroxynon-2-enal.

Oxidative stress enhances lipid peroxidation (LPO) implicated in cancer promotion and progression. (E)-4-Hydroxynon-2-enal 1 (trans-4-hydroxy-2-nonenal, HNE) is one of the most abundant products of LPO. Reactions of HNE with DNA and proteins are responsible for its mutagenic and toxic effects. On the other hand, HNE is regarded as a key molecule in stress mediated cell cycle signaling. LPO generates racemic HNE (rac-1); however, it is expected that the individual enantiomers will behave differently in their interactions with cell components. The study of HNE stereochemistry in its chemical and biochemical interactions is hindered by the lack of expedient methods for preparation of pure enantiomers. This study presents one step synthesis of HNE in a cross-metathesis reaction between the commercially available oct-1-en-3-ol and acrolein in the presence of 2nd generation Grubbs catalyst. The use in the metathesis reaction of enantiomers of oct-1-en-3-ol obtained via Candida antarctica lipase resolution of the racemate allowed us to prepare of 4-(R)- and 4-(S)-enantiomers of HNE (R-1 and S-1, respectively) with excellent optical purity (97.5 and 98.4% ee, respectively) and good chemical yields (70%).

Bacterial DNA repair genes and their eukaryotic homologues: 2. Role of bacterial mutator gene homologues in human disease. Overview of nucleotide pool sanitization and mismatch repair systems.

Since the discovery of the first E. coli mutator gene, mutT, most of the mutations inducing elevated spontaneous mutation rates could be clearly attributed to defects in DNA repair. MutT turned out to be a pyrophosphohydrolase hydrolyzing 8-oxodGTP, thus preventing its incorporation into DNA and suppresing the occurrence of spontaneous AT-->CG transversions. Most of the bacterial mutator genes appeared to be evolutionarily conserved, and scientists were continuously searching for contribution of DNA repair deficiency in human diseases, especially carcinogenesis. Yet a human MutT homologue--hMTH1 protein--was found to be overexpressed rather than inactivated in many human diseases, including cancer. The interest in DNA repair contribution to human diseases exploded with the observation that germline mutations in mismatch repair (MMR) genes predispose to hereditary non-polyposis colorectal cancer (HNPCC). Despite our continuously growing knowledge about DNA repair we still do not fully understand how the mutator phenotype contributes to specific forms of human diseases.

Mismatch dependent uracil/thymine-DNA glycosylases excise exocyclic hydroxyethano and hydroxypropano cytosine adducts.

Exocyclic adducts of DNA bases, such as etheno- and hydroxyalkano- ones, are generated by a variety of bifunctional agents, including endogenously formed products of lipid peroxidation. In this work we selectively modified cytosines in the 5'-d(TTT TTT CTT TTT CTT TTT CTT TTT T)-3' oligonucleotide using: chloroacetaldehyde to obtain 3,N(4)-alpha-hydroxyethano- (HEC) and 3,N(4)-etheno- (epsilonC), acrolein to obtain 3,N(4)-alpha-hydroxypropano- (HPC) and crotonaldehyde to obtain 3,N(4)-alpha-hydroxy-gamma-methylpropano- (mHPC) adducts of cytosine. The studied adducts are alkali-labile which results in oligonucleotide strain breaks at the sites of modification upon strong base treatment. The oligonucleotides carrying adducted cytosines were studied as substrates of Escherichia coli Mug, human TDG and fission yeast Thp1p glycosylases. All the adducts studied are excised by bacterial Mug although with various efficiency: epsilonC >HEC >HPC >mHPC. The yeast enzyme excises efficiently epsilonC>HEC>HPC, whereas the human enzyme excises only epsilonC. The pH-dependence curves of excision of eC, HEC and HPC by Mug are bell shaped and the most efficient excision of adducts occurs within the pH range of 8.6-9.6. The observed increase of excision of HEC and HPC above pH 7.2 can be explained by deprotonation of these adducts, which are high pK(a) compounds and exist in a protonated form at neutrality. On the other hand, since epsilonC is in a neutral form in the pH range studied, we postulate an involvement of an additional catalytic factor. We hypothesize that the enzyme structure undergoes a pH-induced rearrangement allowing the participation of Lys68 of Mug in catalysis via a hydrogen bond interaction of its epsilon-amino group with N(4) of the cytosine exocyclic adducts.

Contribution of hMTH1 to the maintenance of 8-oxoguanine levels in lung DNA of non-small-cell lung cancer patients.

BACKGROUND: The level of 8-oxoguanine (8-oxoG), a general marker of oxidative DNA damage, in DNA is the result of both an equilibrium between the rates of its formation and removal from DNA by DNA repair enzymes and the removal of 8-oxodGTP from the cellular nucleotide pool by hydrolysis to 8-oxodGMP, preventing its incorporation into DNA. To determine the contribution of each component to the level of 8-oxoG in DNA, we compared 8-oxoG-excising activity (encoded by hOGG1), 8-oxodGTPase activity (encoded by hMTH1), and 8-oxoG levels in DNA from tumors and surrounding normal lung tissues from non-small-cell lung cancer patients. METHODS: We measured the level of 8-oxoG in DNA of 47 patients by high-performance liquid chromatography/electrochemical detection (HPLC/ECD), hOGG1 activity in tissue extracts of 56 patients by the nicking assay using an oligodeoxynucleotide containing a single 8-oxoG, and hMTH1 activity in tissue extracts of 33 patients by HPLC/UV detection. All statistical tests were two-sided. RESULTS: The 8-oxoG level was lower in tumor DNA than in DNA from normal lung tissue (geometric mean: 5.81 versus 10.18 8-oxoG/10(6) G, geometric mean of difference = 1.75; P<.001). The hOGG1 activity was also lower in tumor than in normal lung tissue (geometric mean: 8.76 versus 20.91 pmol/h/mg protein, geometric mean of difference = 2.39; P<.001), whereas the hMTH1 activity was higher in tumor than in normal lung tissue (geometric mean: 28.79 versus 8.94 nmol/h/mg protein, geometric mean of difference = 0.31; P<.001). The activity of hMTH1 was three orders of magnitude higher than that of hOGG1 (nanomoles versus picomoles per hour per milligram of protein, respectively). CONCLUSIONS: Several different components contribute to the maintenance of 8-oxoG levels in human DNA, with the greatest contributor being the removal of 8-oxodGTP from the cellular nucleotide pool by hMTH1.

Long-chain adducts of trans-4-hydroxy-2-nonenal to DNA bases cause recombination, base substitutions and frameshift mutations in M13 phage.

Oxidative stress enhances lipid peroxidation (LPO) implicated in the promotion and progression of carcinogenesis. One of the major LPO products is trans-4-hydroxy-2-nonenal (HNE), which was shown to react with guanosine and under peroxidizing conditions also with adenosine. We show here that all four DNA bases are targets for HNE, although displaying different reactivity: dG > dC > dA approximately equal to dT. HPLC and mass spectrometry analyses of HNE reactions with deoxynucleosides showed in each case the formation of several products, with mass peaks corresponding to HNE-dN adducts at a 1:1 and also 2:1 and 3:1 ratios. In the dA, dC and dG reactions, mass peaks corresponding to heptyl-substituted etheno-adducts were also detected, indicating HNE oxidation to its epoxide by air oxygen. In DNA pretreated with HNE, DNA synthesis by T7 DNA polymerase was stopped in a sequence-dependent manner at G > or = C > A and T sites. HNE increased the mutation rates in the lac Z gene of M13 phage transfected into wild type Escherichia coli. The most frequent event was the recombination between lacZ gene sequences in M13 and the E. coli F' factor DNA. Base substitutions and frameshifts were also observed in approximately similar numbers. Over 50% of base substitutions were the C-->T transitions, followed by the G-->C and A-->C transversions. In the E. coli recA strain recombination was not observed, although one mutational G-->T hot-spot appeared within the DNA fragment undergoing recombination in the wild type E. coli. We conclude that long chain HNE adducts to DNA bases arrest DNA synthesis and cause recombination, base substitutions and frameshift mutations in ssDNA.

Neighbouring bases in template influence base-pairing of isoguanine.

Assuming that the efficiency of the incorporation of 5-methyl-2'-deoxyisocytosine-5' triphosphate (dMiCTP) and dTTP opposite isoguanine (iG) is a measure of the proportion of the keto and enol tautomers of iG in the Thermus aquaticus DNA polymerase active centre, we studied the influence of temperature and iG-neighbouring bases in the template on base-pairing of iG. On the basis of experiments with four sequences (3'-TXT-5', 3'-GXG-5', 3'-CXC-5', 3'-CXT-5', where X=iG) at 37, 50, 65 and 80 degrees C, we found that 3'-neighbours decrease the fraction of the keto tautomer in the order C>G>or=T, whereas temperature apparently does not influence the tautomeric equilibrium of iG. The variability of the ratio of incorporation of dMiCTP versus dTTP (5-20) primarily reflects the variability of K (m) values, since V (max) values are roughly similar, which indicates that the iG.MiC and iG.T pairs fit the polymerase active centre equally well. The altering of the base-pairing of iG by sequence context is discussed in relation to tautomerism and miscoding of this oxidized adenine derivative. A key derivative for preparation oligodeoxynucleotides, O (2)-diphenylcarbamoyl- N (6)-[(dimethylamino)ethylidene]-2'-deoxyisoguanosine, is extremely labile (t (1/2)=3.5 min) in 3% trichloroacetic acid/dichloromethane, i.e. under the conditions of automated DNA synthesis, which results in low yield and length heterogeneity of templates.