Uncatalyzed hydrolysis of deoxyuridine, thymidine, and 5-bromodeoxyuridine

Shapiro, Robert; Kang, Sungzong

doi:10.1021/bi00833a004

Cited by 105 publications

(109 citation statements)

References 30 publications

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“…Under these conditions, proton NMR and UV spectroscopic analysis showed that both substrates are converted quantitatively to thymine, with no significant degradation of the pyrimidine ring. The hydrolysis of thymidine to thymine, as indicated by UV analysis, was observed earlier in neutral and acid solution (8). Table 1 summarizes the rate constants, enthalpies, and entropies of activation for diester hydrolysis observed in this study and earlier work.…”

Section: Resultssupporting

confidence: 65%

The time required for water attack at the phosphorus atom of simple phosphodiesters and of DNA

Schroeder

Lad

Wyman

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

243

240

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Phosphodiester linkages, including those that join the nucleotides of DNA, are highly resistant to spontaneous hydrolysis. The rate of water attack at the phosphorus atom of phosphodiesters is known only as an upper limit, based on the hydrolysis of the dimethyl phosphate anion. That reaction was found to proceed at least 99% by C-O cleavage, at a rate suggesting an upper limit of 10 ؊15 s ؊1 for P-O cleavage of phosphodiester anions at 25°C. To evaluate the rate enhancement produced by P-O cleaving phosphodiesterases such as staphylococcal nuclease, we decided to establish the actual value of the rate constant for P-O cleavage of a simple phosphodiester anion. In dineopentyl phosphate, C-O cleavage is sterically precluded so that hydrolysis occurs only by P-O cleavage. Measurements at elevated temperatures indicate that the dineopentyl phosphate anion undergoes hydrolysis in water with a t 1/2 of 30,000,000 years at 25°C, furnishing an indication of the resistance of the internucleotide linkages of DNA to water attack at phosphorus. These results imply that staphylococcal nuclease (k cat ‫؍‬ 95 s ؊1 ) enhances the rate of phosphodiester hydrolysis by a factor of Ϸ10 17 . In alkaline solution, thymidylyl-3 -5 -thymidine (TpT) has been reported to decompose 10 5 -fold more rapidly than does dineopentyl phosphate. We find however that TpT and thymidine decompose at similar rates and with similar activation parameters, to a similar set of products, at pH 7 and in 1 M KOH. We infer that the decomposition of TpT is initiated by the breakdown of thymidine, not by phosphodiester hydrolysis.DNA hydrolysis ͉ DNA stability ͉ nuclease ͉ rate enhancement ͉ phosphate ester P hosphoric acid diesters are, in general, exceedingly unreactive in water (1-3), so that the phosphodiester linkages that join the nucleotides of DNA are highly resistant to spontaneous hydrolysis. By extrapolation of earlier model experiments at elevated temperatures, the uncatalyzed hydrolysis of dimethyl phosphate in neutral solution was found to proceed with an estimated rate constant of Ϸ2 ϫ 10 Ϫ13 s Ϫ1 at 25°C, corresponding to a half-time of 140,000 years. That reaction was found to proceed at least 99% by C-O cleavage, suggesting an upper limit of Ϸ1 ϫ 10 Ϫ15 s Ϫ1 at 25°C on the rate constant for spontaneous P-O cleavage of a phosphodiester anion, the reaction that is catalyzed by many phosphodiesterases (4).More recently, a rate constant of 6 ϫ 10 Ϫ7 s Ϫ1 has been reported for the decomposition of thymidylyl-3Ј-5Ј-thymidine (TpT) at 80°C in 1 M KOH (5). Extrapolation of the results obtained earlier for dimethyl phosphate hydrolysis in neutral solution (4), to 80°C, would indicate a rate Ϸ10 5 -fold slower. That discrepancy might indicate a major role for catalysis by hydroxide, but the hydrolysis of another dialkyl phosphodiester, bis-3-(4-carboxyphenyl)neopentyl phosphate (Np* 2 P), in which ␥-branching of the leaving alcohol prevents C-O cleavage (Fig. 1), also proceeds Ϸ10 5 -fold more slowly in 1 M KOH (6).In an effort to resolve that discre...

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Section: Resultssupporting

confidence: 65%

The time required for water attack at the phosphorus atom of simple phosphodiesters and of DNA

Schroeder

Lad

Wyman

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

243

240

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“…44,56 Indeed, the N1 and N3 acidities of uracil differ by 14 kcal/mol in the gas phase, 44,46,56 whereas they are nearly equivalent in aqueous solution. 22 Thus, as indicated by previous studies, gas-phase acidities can be informative regarding the leaving ability of a nucleobase within a hydrophobic active site, and may be relevant to the mechanism of substrate discrimination. 44,71 We therefore examined the dependence of hTDG activity on N1 acidity in a non-polar environment by calculating the gas phase N1 acidities of the nucleobases at 298 K (ΔG acid,g , Table S1, Supporting Information).…”

Section: Linear Free Energy Relationships For N-glycosidic Bond Hydromentioning

confidence: 85%

“…A plot of log(k non ) versus pK a N1 for the spontaneous hydrolysis of dU, dT, and 5-Br-dU at pH 6.5 and 75 °C has a slope of β lg = −0.86 ± 0.03, 22,75 and the same value (β lg = −0.86 ± 0.05) is obtained using k non values extrapolated to 22 °C for dU, dT, 5-F-dU, 5-Cl-dU, and 5-Br-dU ( Figure S3, Supporting Information). These β lg values, together with the observed low and positive activation entropies, 22 are consistent with a highly dissociative transition state for the nonenzymatic reaction, 22,23,76 although it was concluded from computational studies that the mechanism is concerted (A N D N ) rather than stepwise (D N *A N ). 77 The more negative β lg = −1.6 observed for hTDG indicates that the transition state (TS) of the enzymatic reaction is more sensitive to the development of negative charge on N1 of the leaving group base than is the TS of the non-enzymatic reaction.…”

Section: Implications Of the Brønsted-type Lfer For The Mechanism Of mentioning

confidence: 99%

Specificity of Human Thymine DNA Glycosylase Depends on N-Glycosidic Bond Stability

et al. 2006

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Initiating the DNA base excision repair pathway, DNA glycosylases find and hydrolytically excise damaged bases from DNA. While some DNA glycosylases exhibit narrow specificity, others remove multiple forms of damage. Human thymine DNA glycosylase (hTDG) cleaves thymine from mutagenic G·T mispairs and recognizes many additional lesions, and has a strong preference for nucleobases paired with guanine rather than adenine. Yet, hTDG avoids cytosine, despite the millionfold excess of normal G·C pairs over G·T mispairs. The mechanism of this remarkable and essential specificity has remained obscure. Here, we examine the possibility that hTDG specificity depends on the stability of the scissile base-sugar bond by determining the maximal activity (k max ) against a series of nucleobases with varying leaving group ability. We find that hTDG removes 5-fluorouracil 78-fold faster than uracil and 5-chlorouracil 572-fold faster than thymine, differences that can be attributed predominantly to leaving group ability. Moreover, hTDG readily excises cytosine analogues with improved leaving ability, including 5-fluorocytosine, 5-bromocytosine, and 5-hydroxycytosine, indicating that cytosine has access to the active site. A plot of log(k max ) versus leaving group pK a reveals a Brønsted-type linear free energy relationship with a large negative slope of β lg = −1.6 ± 0.2, consistent with a highly dissociative reaction mechanism. Further, we find that the hydrophobic active site of hTDG contributes to its specificity by enhancing the inherent differences in substrate reactivity. Thus, hTDG specificity depends on N-glycosidic bond stability, and the discrimination against cytosine is due largely to its very poor leaving ability rather than its exclusion from the active site.

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“…The substrate reactivity (N-glycosidic bond stability) also depends on the C5 substituent. Thus, for the hTDG-catalyzed and non-catalyzed reactions, the rate depends on the leaving ability of the departing nucleobase (20,(42)(43)(44). The electron withdrawing halogens enhance the leaving ability of uracil, whereas the electron donating methyl group suppresses it.…”

Section: Discussionmentioning

confidence: 99%

Excision of 5-Halogenated Uracils by Human Thymine DNA Glycosylase

Morgan

Bennett

Drohat

2007

Journal of Biological Chemistry

200

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Thymine DNA glycosylase (TDG) excises thymine from G⅐T mispairs and removes a variety of damaged bases (X) with a preference for lesions in a CpG⅐X context. We recently reported that human TDG rapidly excises 5-halogenated uracils, exhibiting much greater activity for CpG⅐FU, CpG⅐ClU, and CpG⅐BrU than for CpG⅐T. Here we examine the effects of altering the CpG context on the excision activity for U, T, FU, ClU, and BrU. We show that the maximal activity (k max ) for G⅐X substrates depends significantly on the 5 base pair. For example, k max decreases by 6-, 11-, and 82-fold for TpG⅐ClU, GpG⅐ClU, and ApG⅐ClU, respectively, as compared with CpG⅐ClU. For the other G⅐X substrates, the 5-neighbor effects have a similar trend but vary in magnitude. The activity for G⅐FU, G⅐ClU, and G⅐BrU, with any 5-flanking pair, meets and in most cases significantly exceeds the CpG⅐T activity. Strikingly, human TDG activity is reduced 10 2.3 -10 4.3 -fold for A⅐X relative to G⅐X pairs and reduced further for A⅐X pairs with a 5 pair other than C⅐G. The effect of altering the 5 pair and/or the opposing base (G⅐X versus A⅐X) is greater for substrates that are larger (bromodeoxyuridine, dT) or have a more stable N-glycosidic bond (such as dT). The largest CpG context effects are observed for the excision of thymine. The potential role played by human TDG in the cytotoxic effects of ClU and BrU incorporation into DNA, which can occur under inflammatory conditions and in the cytotoxicity of FU, a widely used anticancer agent, are discussed.The nucleobases in DNA are subject to continuous chemical modification, generating a broad range of mutagenic and cytotoxic lesions that can lead to cancer and other diseases (1, 2). To counteract this inevitable damage, the cellular machinery includes systems for DNA repair (3). Damage occurring to the nucleobases is the purview of base excision repair, a pathway that is initiated by a damage-specific DNA glycosylase. These enzymes find damaged or mismatched bases within the vast expanse of normal DNA and catalyze the cleavage of the basesugar (N-glycosidic) bond, producing an abasic or apurinic/ apyrimidinic (AP) 2 site in the DNA. The repair process is continued by follow-on base excision repair enzymes.Human thymine DNA glycosylase (hTDG) was discovered as an enzyme that removes thymine from G⅐T and uracil from G⅐U mispairs in DNA (4, 5). In vertebrates, G⅐T mispairs arise from replication errors, which are handled by the mismatch repair pathway or from the deamination of 5-methylcytosine to T (6, 7). Because cytosine methylation occurs at CpG dinucleotides (8, 9), G⅐T mispairs caused by 5-methylcytosine deamination are found at CpG sites. It has been shown that hTDG is most active for G⅐T mispairs with a 5Ј C⅐G pair, suggesting that a predominant biological role of the enzyme is to initiate the repair of CpG⅐T lesions (10, 11). DNA methylation at CpG plays a fundamental role in many cellular processes, including transcriptional regulation and the silencing of repetitive genetic elements (8, 9). Sugg...

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Uncatalyzed hydrolysis of deoxyuridine, thymidine, and 5-bromodeoxyuridine

Cited by 105 publications

References 30 publications

The time required for water attack at the phosphorus atom of simple phosphodiesters and of DNA

The time required for water attack at the phosphorus atom of simple phosphodiesters and of DNA

Specificity of Human Thymine DNA Glycosylase Depends on N-Glycosidic Bond Stability

Excision of 5-Halogenated Uracils by Human Thymine DNA Glycosylase

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