Specificity of human thymine DNA glycosylase depends on N-glycosidic bond stability - PubMed (original) (raw)

Specificity of human thymine DNA glycosylase depends on N-glycosidic bond stability

Matthew T Bennett et al. J Am Chem Soc. 2006.

Abstract

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, recognizes many additional lesions, and has a strong preference for nucleobases paired with guanine rather than adenine. Yet, hTDG avoids cytosine, despite the million-fold 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 beta(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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Figures

Figure 1

Known hTDG substrates are shown.– Cytosine and 5-methyl-C are not significant substrates., We report several new hTDG substrates here, including 5-chloro-U, 5-iodo-U, 5-fluoro-C, and 5-bromo-C.

Figure 2

HPLC assay for monitoring DNA glycosylase reactions. Shown are chromatograms for samples taken from a single-turnover reaction at an “early” time point where there was little product formation, and a “late” time point where the reaction was nearly complete. The ion-exchange HPLC under denaturing (pH 12) conditions gives excellent resolution of the target strand, its complement, and the two shorter product strands resulting from alkaline-induced cleavage of the nascent abasic strand produced by hTDG activity.

Figure 3

A minimal kinetic mechanism for the hTDG-catalyzed reaction. The initial association of hTDG (E) and substrate DNA (D) produces the collision complex (E·D) followed by a base-flipping step to form the reactive complex (E·BD, where BD is DNA with an extrahelical base). Cleavage of the base-sugar bond (_k_chem) yields the ternary product complex (E·B·apD). Release of the base (B) likely precedes very slow release of AP DNA (apD)., The _k_max values report on the reaction steps from the initial hTDG·DNA collision complex to the ternary product complex.

Figure 4

Single turnover kinetics for hTDG. (A) Structure of the 19 bp DNA substrate used for the kinetics experiments, where the target base (x) is located at a CpG site (bold and underlined). A substrate in which x = uracil is referred to as G·U19 (B) Representative data for the maximal activity of hTDG (5 µM) against G·U19 (500 nM) gives a rate constant of _k_max = 2.6 min−1. Data for G·U19 were collected using manual sampling and a rapid chemical quench-flow instrument (for t <8 s). (C) Confirming the saturating enzyme conditions, _k_max values for G·T19 are independent of enzyme for hTDG concentrations ≥ 1.0 µM: _k_obs = 0.20 min−1 for 1.0 µM hTDG (●); _k_obs = 0.21 min−1 for 2.5 µM hTDG (○); _k_obs = 0.23 min−1 for 5 µM hTDG (▼); _k_obs = 0.22 min−1 for 10 µM hTDG (□).

Figure 5

Electrostatic potential maps of the 5-substituted uracil and cytosine bases used in this work. The range of electrostatic potential varies between −31 kcal/mol (red) to +31 kcal/mol (blue), on an isosurface of 0.002 electrons/Å3 of the total SCF electron density.

Figure 6

Representative data for the activity of hTDG against 5-halouracils. (A) hTDG rapidly excises FU, _k_max = 202 min−1 (●), and ClU, _k_max = 126 min−1 (▼). (B) The removal of BrU is significantly slower, _k_max = 11.6 min−1, likely due to steric effects with the active site (see text).

Figure 7

hTDG excises cytosine analogs with improved leaving group ability. Shown are representative data from single turnover experiments for (A) the removal of FC from G·FC pairs, _k_max = 0.035 min−1, and (B) the removal of BrC from G·BrC pairs, _k_max = 0.008 min−1 (●), and hoC from G·hoC pairs, _k_max = 0.010 min−1 (▼).

Figure 8

Brønsted-type linear free energy relationship (LFER) for the hTDG reaction. The LFER has a good correlation coefficient (r = 0.96) and a large negative slope of βlg = −1.6 ± 0.2 (Brønsted coefficient). The LFER includes data for U, T, FU, ClU, hoU, hmU, FC, hoC, and C (○) and covers a range in leaving group p_K_aN1 of >4 log units. A determination of the LFER using only the 5-substituted uracils gives essentially the same result, βlg = −1.4 ± 0.2, r = 0.96. Data for BrU, IU, and BrC are shown (□) but were not included in the LFER because the _k_max values for these bases suggests limited access to the active site (see text).

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Specificity of human thymine DNA glycosylase depends on N-glycosidic bond stability - PubMed (original) (raw)