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

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

Comparison of the Structural and Dynamic Effects of 5‑Methylcytosine and 5‑Chlorocytosine in a CpG Dinucleotide Sequence

Inflammation-mediated reactive molecules can result in an array of oxidized and halogenated DNA-damage products, including 5-chlorocytosine (^ClC). Previous studies have shown that ^ClC can mimic 5-methylcytosine (^mC) and act as a fraudulent epigenetic signal, promoting the methylation of previously unmethylated DNA sequences. Although the 5-halouracils are good substrates for base-excision repair, no repair activity has yet been identified for ^ClC. Because of the apparent biochemical similarities of ^mC and ^ClC, we have investigated the effects of ^mC and ^ClC substitution on oligonucleotide structure and dynamics. In this study, we have constructed oligonucleotide duplexes containing C, ^ClC, and ^mC within a CpG dinucleotide. The thermal and thermodynamic stability of these duplexes were found to be experimentally indistinguishable. Crystallographic structures of duplex oligonucleotides containing ^mC or ^ClC were determined to 1.2 and 1.9 Å resolution, respectively. Both duplexes are B-form and are superimposable on a previously determined structure of a cytosine-containing duplex with a rmsd of approximately 0.25 Å. NMR solution studies indicate that all duplexes containing cytosine or the cytosine analogues are normal B-form and that no structural perturbations are observed surrounding the site of each substitution. The magnitude of the base-stacking-induced upfield shifts for nonexchangeable base proton resonances are similar for each of the duplexes examined, indicating that neither ^mC nor ^ClC significantly alter base-stacking interactions. The ^ClC analogue is paired with G in an apparently normal geometry; however, the G-imino proton of the ^ClC–G base pair resonates to higher field relative to ^mC–G or C–G, indicating a weaker imino hydrogen bond. Using selective ¹⁵N-enrichment and isotope-edited NMR, we observe that the amino group of ^ClC rotates at roughly half of the rate of the corresponding amino groups of the C–G and ^mC–G base pairs. The altered chemical shifts of hydrogen-bonding proton resonances for the ^ClC–G base pair as well as the slower rotation of the ^ClC amino group can be attributed to the electron-withdrawing inductive property of the 5-chloro substituent. The apparent similarity of duplexes containing ^mC and ^ClC demonstrated here is in accord with results of previous biochemical studies and further suggests that ^ClC is likely to be an unusually persistent form of DNA damage