Mechanisms of base selection by the E.coli mispaired uracil glycosylase

The repair of the multitude of single-base lesions formed daily in the cells of all living organisms is accomplished primarily by the base-excision repair (BER) pathway that initiates repair through a series of lesion-selective glycosylases. In this paper, single-turnover kinetics have been measured on a series of oligonucleotide substrates containing both uracil and purine analogs for the E. coli mispaired uracil glycosylase, MUG. The relative rates of glycosylase cleavage have been correlated with the free energy of helix formation, and with the size and electronic inductive properties of a series of uracil 5-substituents. Data is presented that MUG can exploit the reduced thermodynamic stability of mispairs to distinguish U:A from U:G pairs. Discrimination against the removal of thymine results primarily from the electron-donating property of the thymine 5-methyl substituent, while the size of the methyl group relative to a hydrogen atom is a secondary factor. A series of parameters have been obtained that allow prediction of relative MUG cleavage rates that correlate well with observed relative rates that vary over five orders of magnitude for the series of base analogs examined. We propose that these parameters may be common among DNA glycosylases, however, specific glycosylases may focus more or less on each of the parameters identified. The presence of a series of glycosylases which focus on different lesion properties, all coexisting within the same cell, would provide a robust and partially redundant repair system necessary for the maintenance of the genome

Polyamines stimulate the formation of mutagenic 1,N(2)-propanodeoxyguanosine adducts from acetaldehyde

Alcoholic beverage consumption is associated with an increased risk of upper gastrointestinal cancer. Acetaldehyde (AA), the first metabolite of ethanol, is a suspected human carcinogen, but the molecular mechanisms underlying AA carcinogenicity are unclear. In this work, we tested the hypothesis that polyamines could facilitate the formation of mutagenic α-methyl-γ-hydroxy-1,N(2)-propano-2′-deoxyguanosine (Cr-PdG) adducts from biologically relevant AA concentrations. We found that Cr-PdG adducts could be formed by reacting deoxyguanosine with μM concentrations of AA in the presence of spermidine, but not with either AA or spermidine alone. The identities of the Cr-PdG adducts were confirmed by both liquid and gas chromatography-mass spectrometry. Using a novel isotope-dilution liquid chromatography-mass spectrometry assay, we found that in the presence of 5 mM spermidine, AA concentrations of 100 μM and above resulted in the formation of Cr-PdG in genomic DNA. These AA levels are within the range that occurs in human saliva after alcoholic beverage consumption. We also showed that spermidine directly reacts with AA to generate crotonaldehyde (CrA), most likely via an enamine aldol condensation mechanism. We propose that AA derived from ethanol metabolism is converted to CrA by polyamines in dividing cells, forming Cr-PdG adducts, which may be responsible for the carcinogenicity of alcoholic beverage consumption

Mechanisms of base selection by human single-stranded selective monofunctional uracil-DNA glycosylase

hSMUG1 (human single-stranded selective monofunctional uracil-DNA glyscosylase) is one of three glycosylases encoded within a small region of human chromosome 12. Those three glycosylases, UNG (uracil-DNA glycosylase), TDG (thymine-DNA glyscosylase), and hSMUG1, have in common the capacity to remove uracil from DNA. However, these glycosylases also repair other lesions and have distinct substrate preferences, indicating that they have potentially redundant but not overlapping physiological roles. The mechanisms by which these glycosylases locate and selectively remove target lesions are not well understood. In addition to uracil, hSMUG1 has been shown to remove some oxidized pyrimidines, suggesting a role in the repair of DNA oxidation damage. In this paper, we describe experiments in which a series of oligonucleotides containing purine and pyrimidine analogs have been used to probe mechanisms by which hSMUG1 distinguishes potential substrates. Our results indicate that the preference of hSMUG1 for mispaired uracil over uracil paired with adenine is best explained by the reduced stability of a duplex containing a mispair, consistent with previous reports with Escherichia coli mispaired uracil-DNA glycosylase. We have also extended the substrate range of hSMUG1 to include 5-carboxyuracil, the last in the series of damage products from thymine methyl group oxidation. The properties used by hSMUG1 to select damaged pyrimidines include the size and free energy of solvation of the 5-substituent but not electronic inductive properties. The observed distinct mechanisms of base selection demonstrated for members of the uracil glycosylase family help explain how considerable diversity in chemical lesion repair can be achieved

Real-time detection of Fe·EDTA/H2O2-induced DNA cleavage by linear dichroism

The conditions for the measurement of linear dichroism (LD) can be adjusted so as to solely reflect the length and the flexibility of DNA. The real-time detection of the EDTA·Fe2+-induced oxidative cleavage of double-stranded native and synthetic DNAs was performed using LD. The decrease in the magnitude of the LD at 260 nm, which reflects an increase in the flexibility and a decrease in the length of the DNA, can be described by the sum of two or three exponential curves in relation to the EDTA·Fe2+ concentration. The fast component was assigned to the cleavage of one of the double strands, inducing an increase in the flexibility, while the other slower component was assigned to the cleavage of the double strand, resulting in the shortening of DNA. The decrease in the magnitude of the LD of poly[d(A-T)2] was similar to that of poly[d(I-C)2], while that of poly[d(G-C)2] was found to be the slowest, indicating that the resistance of poly[d(G-C)2] against the Fenton-type reagent was the strongest. This observation suggests that the amine group in the minor groove of the double helix may play an important role in slowing the EDTA·Fe2+-induced oxidative cleavage

Short-term salivary acetaldehyde increase due to direct exposure to alcoholic beverages as an additional cancer risk factor beyond ethanol metabolism

<p>Abstract</p> <p>Background</p> <p>An increasing body of evidence now implicates acetaldehyde as a major underlying factor for the carcinogenicity of alcoholic beverages and especially for oesophageal and oral cancer. Acetaldehyde associated with alcohol consumption is regarded as 'carcinogenic to humans' (IARC Group 1), with sufficient evidence available for the oesophagus, head and neck as sites of carcinogenicity. At present, research into the mechanistic aspects of acetaldehyde-related oral cancer has been focused on salivary acetaldehyde that is formed either from ethanol metabolism in the epithelia or from microbial oxidation of ethanol by the oral microflora. This study was conducted to evaluate the role of the acetaldehyde that is found as a component of alcoholic beverages as an additional factor in the aetiology of oral cancer.</p> <p>Methods</p> <p>Salivary acetaldehyde levels were determined in the context of sensory analysis of different alcoholic beverages (beer, cider, wine, sherry, vodka, calvados, grape marc spirit, tequila, cherry spirit), without swallowing, to exclude systemic ethanol metabolism.</p> <p>Results</p> <p>The rinsing of the mouth for 30 seconds with an alcoholic beverage is able to increase salivary acetaldehyde above levels previously judged to be carcinogenic in vitro, with levels up to 1000 μM in cases of beverages with extreme acetaldehyde content. In general, the highest salivary acetaldehyde concentration was found in all cases in the saliva 30 sec after using the beverages (average 353 μM). The average concentration then decreased at the 2-min (156 μM), 5-min (76 μM) and 10-min (40 μM) sampling points. The salivary acetaldehyde concentration depends primarily on the direct ingestion of acetaldehyde contained in the beverages at the 30-sec sampling, while the influence of the metabolic formation from ethanol becomes the major factor at the 2-min sampling point.</p> <p>Conclusions</p> <p>This study offers a plausible mechanism to explain the increased risk for oral cancer associated with high acetaldehyde concentrations in certain beverages.</p

Complete release of (5′S)-8,5′-cyclo-2′-deoxyadenosine from dinucleotides, oligodeoxynucleotides and DNA, and direct comparison of its levels in cellular DNA with other oxidatively induced DNA lesions

8,5′-Cyclopurine-2′-deoxynucleosides in DNA are repaired by nucleotide-excision repair, and act as strong blocks to DNA polymerases, RNA polymerase II and transcription factor binding. Thus, it is important to accurately determine the level of these lesions in DNA. There is controversy in the literature regarding the ability of different enzymes to release these compounds from oligodeoxynucleotides or DNA. We used liquid chromatography/mass spectrometry (LC/MS) to investigate the ability of several enzymes to release (5′S)-8,5′-cyclo-2′-deoxyadenosine [(5′S)-cdA] from dinucleotides and oligodeoxynucleotides and from DNA. The data show that (5′S)-cdA is completely released from DNA by hydrolysis with nuclease P1, snake venom phosphodiesterase and alkaline phosphatase. The identity of the normal nucleoside 5′ to the (5′S)-cdA had a significant effect on its release. Using LC/MS, we also showed that the levels of (5′S)-cdA were within an order of magnitude of those of 8-hydroxy-2′-deoxyguanosine, and three times higher than those of 8-hydroxy-2′-deoxyadenosine in pig liver DNA. Different DNA isolation methods affected the levels of the latter two lesions, but did not influence those of (5′S)-cdA. We conclude that (5′S)-cdA can be completely released from DNA by enzymic hydrolysis, and the level of (5′S)-cdA in tissue DNA is comparable to those of other oxidatively induced DNA lesions

The reactions of thymine and thymidine with ozone

The ozonolysis of thymine and thymidine has been investigated by a product study complemented by kinetic studies using spectrophotometry, conductometry and stopped-flow with optical and conductometric detection. Material balance has been obtained. Ozonolysis of thymine (k = 3.4 x 10(4) dm(3) mol(-1) s(-1)) leads to the formation of the acidic (pK(a) = 4) hydroperoxide 1-hydroperoxymethylene-3-(2-oxopropanoyl)urea 5 (similar to34%), neutral hydroperoxides (possibly mainly 1- hydroperoxyhydroxymethyl-3-(2-oxopropanoyl)urea 6, total similar to41%) and H2O2 (25%, with corresponding formation of 1-formyl-5-hydroxy-5-methylhydantoin 11). The organic hydroperoxides decay (similar to1.1 x 10(-3) s(-1) at 20 degreesC, 1.3 x 10(-4) s(-1) at 3 degreesC) releasing formic acid (formation of 5-hydroperoxy-5-methylhydantoin 18) and also to some extent H2O2 (and 11). After 100 min, the formic acid yield is 75%. Upon treatment at high pH, it increases to 100%. Reduction of the organic hydroperoxides with bis(2- hydroxyethyl)sulfide (k = 50 dm(3) mol(-1) s(-1)) leads to 11 whose subsequent treatment with base yields 5-hydroxy-5- methylhydantoin 13 in 100% yield. It is suggested that the Criegee ozonide formed upon reaction with ozone at the C(5)- C(6) double bond opens heterolytically in two directions with subsequent opening of the C(5)-C( 6) bond. In the preferred route (75%), the positive charge resides at C(6). Deprotonation at N(1) gives rise to 5, while its reaction with water yields 6. Loss of formic acid yields 5-hydroperoxy-5- methylhydantoin 18. Reduction of 5 and 6 with the sulfide yields 11. In the minor route (25%), the positive charge remains at C(5) followed by a reaction with water. The resulting alpha-hydroxy hydroperoxide rapidly loses H2O2 (formation of 11). In basic solution, singlet dioxygen is formed (8%). The concomitant product, 5,6-dihydroxy-5,6-dihydrothymine has been detected. In the ozonolysis of thymidine, the rapid formation of conductance (k = 0.55 s(-1)) is due to the release of acetic acid (18%). In this reaction a short-lived hydroperoxide is destroyed. As a consequence of this, 25 s after ozonolysis the total hydroperoxide yield is only 78% (including 8% H2O2). The products corresponding to acetic acid are suggested to be CO2 and N-(2-deoxy-beta-D-erythropentofuranosyl)formylurea 22. A number of organic hydroperoxides have been detected by HPLC by post-column derivatisation with iodide. An acidic hydroperoxide such as 5 in the case of thymine is not among the products. Upon sulfide reduction, the organic hydroperoxides yield mainly (43-50%) N-1-(2-deoxy-beta-D-erythropentofuranosyl)-5-hydroxy- 5-methylhydantoin 23. The reasons for some striking differences in the ozonolyses of thymine and thymidine are discussed

1,N 2-propanodeoxyguanosine adducts from

acetaldehyd

Enhancement of camptothecin-induced topoisomerase I cleavage complexes by the acetaldehyde adduct N(2)-ethyl-2′-deoxyguanosine

The activity of DNA topoisomerase I (Top1), an enzyme that regulates DNA topology, is impacted by DNA structure alterations and by the anticancer alkaloid camptothecin (CPT). Here, we evaluated the effect of the acetaldehyde-derived DNA adduct, N(2)-ethyl-2′-deoxyguanosine (N(2)-ethyl-dG), on human Top1 nicking and closing activities. Using purified recombinant Top1, we show that Top1 nicking-closing activity remains unaffected in N(2)-ethyl-dG adducted oligonucleotides. However, the N(2)-ethyl-dG adduct enhanced CPT-induced Top1–DNA cleavage complexes depending on the relative position of the N(2)-ethyl-dG adduct with respect to the Top1 cleavage site. The Top1-mediated DNA religation (closing) was selectively inhibited when the N(2)-ethyl-dG adduct was present immediately 3′ from the Top1 site (position +1). In addition, when the N(2)-ethyl-dG adduct was located at the −5 position, CPT enhanced cleavage at an alternate Top1 cleavage site immediately adjacent to the adduct, which was then at position +1 relative to this new alternate Top1 site. Modeling studies suggest that the ethyl group on the N(2)-ethyl-dG adduct located at the 5′ end of a Top1 site (position +1) sterically blocks the dissociation of CPT from the Top1–DNA complex, thereby inhibiting further the religation (closing) reaction