The three-dimensional structure of Ca2+-bound rat S100B(ββ) has been determined using data from a series of two-dimensional (2D), three-dimensional (3D), and four-dimensional (4D) nuclear magnetic ...resonance (NMR) experiments. Each S100β subunit (91 residues) contains four helixes (helix 1, E2−R20; helix 2, K29−N38; helix 3, Q50−D61; and helix 4, F70−A83) and one antiparallel β-sheet (strand 1, K26−K28; and strand 2, E67−D69) which brings the normal and pseudo EF-hands together. As found previously for rat apo-S100B(ββ) Drohat, A. C., et al. (1996) Biochemistry 35, 11577−11588, helixes 1, 1‘, 4, and 4‘ associate to form an X-type four-helix bundle at the symmetric dimer interface. Additionally, Ca2+ binding does not significantly change the interhelical angle of helixes 1 and 2 in the pseudo EF-hand (apo, Ω1 - 2 = 132 ± 4°; and Ca2+-bound, Ω1 - 2 = 137 ± 5°). However, the interhelical angle of helixes 3 and 4 in the normal EF-hand (Ω3 - 4 = 106 ± 4°) changed significantly upon the addition of Ca2+ (ΔΩ3 - 4 = 112 ± 5°) and is similar to that of the Ca2+-bound EF-hands in calbindin D9K, calmodulin, and troponin (84° ≤ Ω ≤ 128°). Further, the four helixes within each S100β subunit form a splayed-type four-helix bundle (four perpendicular helixes) as observed in Ca2+-bound calbindin D9K. The large Ca2+-dependent conformational change involving helix 3 exposes a cleft, defined by residues in the hinge region, the C-terminal loop, and helix 3, which is absent in the apo structure. This surface on Ca2+-bound S100B(ββ) is likely important for target protein binding.
Escherichia coli uracil DNA glycosylase (UDG) catalyzes the hydrolysis of premutagenic uracil bases in DNA by flipping the deoxyuridine from the DNA helix Stivers, J. T., et al. (1999) Biochemistry ...38, 952. A general acid−base mechanism has been proposed whereby His187 facilitates leaving group departure by protonating the O2 of uracil and Asp64 activates a water molecule for nucleophilic attack at C1‘ of the deoxyribose. Detailed kinetic studies on the H187Q, H187A, and D64N mutant enzymes indicate that Asp64 and His187 stabilize the chemical transition state by 5.3 and 4.8 kcal/mol, respectively, with little effect on substrate or product binding. The pH dependence of k cat for wild-type and H187Q UDG indicates that an unprotonated group in the enzyme−substrate complex (pK a = 6.2 ± 0.2) is required for catalysis. This unprotonated group has a small ΔH of ionization (−0.4 ± 1.7 kcal/mol) and is absent in the pH profile for D64N UDG, suggesting that it corresponds to the general base Asp64. The pH dependence of k cat for wild-type, H187Q, and D64N UDG shows no evidence for an essential protonated group over the pH range of 5.5−10. Hence, the pK a of His187 must be outside this pH range if it serves as an electrophilic catalyst. These results support a mechanism in which Asp64 serves as the general base and His187 acts as a neutral electrophile, stabilizing a developing negative charge on uracil O2 in the transition state. In the following paper of this issue we establish by crystallography and heteronuclear NMR spectroscopy that the imidazole of His187 is neutral during the catalytic cycle of UDG.
Human (h) DNA repair enzyme thymine DNA glycosylase (hTDG) is a key DNA glycosylase in the base excision repair (BER) pathway that repairs deaminated cytosines and 5-methyl-cytosines. The cell cycle ...checkpoint protein Rad9-Rad1-Hus1 (the 9-1-1 complex) is the surveillance machinery involved in the preservation of genome stability. In this study, we show that hTDG interacts with hRad9, hRad1 and hHus1 as individual proteins and as a complex. The hHus1 interacting domain is mapped to residues 67-110 of hTDG, and Val74 of hTDG plays an important role in the TDG-Hus1 interaction. In contrast to the core domain of hTDG (residues 110-308), hTDG(67-308) removes U and T from U/G and T/G mispairs, respectively, with similar rates as native hTDG. Human TDG activity is significantly stimulated by hHus1, hRad1, hRad9 separately, and by the 9-1-1 complex. Interestingly, the interaction between hRad9 and hTDG, as detected by co-immunoprecipitation (Co-IP), is enhanced following N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) treatment. A significant fraction of the hTDG nuclear foci co-localize with hRad9 foci in cells treated with methylating agents. Thus, the 9-1-1 complex at the lesion sites serves as both a damage sensor to activate checkpoint control and a component of the BER.
The specific recognition mechanisms of DNA repair glycosylases that remove cationic alkylpurine bases in DNA are not well
understood partly due to the absence of structures of these enzymes with ...their cognate bases. Here we report the solution
structure of 3-methyladenine DNA glycosylase I (TAG) in complex with its 3-methyladenine (3-MeA) cognate base, and we have
used chemical perturbation of the base in combination with mutagenesis of the enzyme to evaluate the role of hydrogen bonding
and Ï-cation interactions in alkylated base recognition by this DNA repair enzyme. We find that TAG uses hydrogen bonding
with heteroatoms on the base, van der Waals interactions with the 3-Me group, and conventional Ï-Ï stacking with a conserved
Trp side chain to selectively bind neutral 3-MeA over the cationic form of the base. Discrimination against binding of the normal base adenine is derived from direct
sensing of the 3-methyl group, leading to an induced-fit conformational change that engulfs the base in a box defined by five
aromatic side chains. These findings indicate that base specific recognition by TAG does not involve strong Ï-cation interactions,
and suggest a novel mechanism for alkylated base recognition and removal.
Uracil DNA glycosylase (UDG) cleaves the glycosidic bond of deoxyuridine in DNA using a hydrolytic mechanism, with an overall catalytic rate enhancement of 1012-fold over the solution reaction. The ...nature of the enzyme−substrate interactions that lead to this large rate enhancement are key to understanding enzymatic DNA repair. Using 1H and heteronuclear NMR spectroscopy, we have characterized one such interaction in the ternary product complex of Escherichia coli UDG, the short (2.7 Å) H bond between His187 Nε2 and uracil O2. The H bond proton is highly deshielded at 15.6 ppm, indicating a short N-O distance and exhibits a solvent exchange rate that is 400- and 105-fold slower than free imidazole at pH 7.5 and pH 10, respectively. Heteronuclear NMR experiments at neutral pH show that this H bond involves the neutral imidazole form of His187 and the N1-O2 imidate form of uracil. The excellent correspondence of the pK a for the disappearance of the H bond (pK a = 6.3 ± 0.1) with the previously determined pK a = 6.4 for the N1 proton of enzyme-bound uracil indicates that the H bond requires negative charge on uracil O2 Drohat, A. C., and Stivers, J. T. (2000) J. Am. Chem. Soc. 122, 1840−1841. Although the above characteristics suggest a short strong H bond, the D/H fractionation factor of φ = 1.0 is more typical of a normal H bond. This unexpected observation may reflect a large donor−acceptor pK a mismatch or the net result of two opposing effects on vibrational frequencies: decreased N-H bond stretching frequencies (φ < 1) and increased bending frequencies (φ > 1) relative to the O-H bonds of water. The role of this H bond in catalysis by UDG and several approaches to quantify the H bond energy are discussed.
The DNA repair enzyme uracil DNA glycosylase (UDG) hydrolyzes the glycosidic bond of deoxyuridine in DNA by a remarkable mechanism
involving formation of a positively charged oxacarbenium ion-uracil ...anion intermediate. We have proposed that the positively
charged intermediate is stabilized by being sandwiched between the combined negative charges of the anionic uracil leaving
group and a conserved aspartate residue that are located on opposite faces of the sugar ring. Here we establish that a duplex
DNA oligonucleotide containing a cationic 1-aza-deoxyribose (I) oxacarbenium ion mimic is a potent inhibitor of UDG that binds
tightly to the enzyme-uracil anion (EU â ) product complex ( K
D of EU â = 110 p m ). The tight binding of I to the EU â complex results from its extremely slow off rate ( k
off = 0.0008 s â1 ), which is 25,000-fold slower than substrate analogue DNA. Removal of Asp 64 and His 187 , which are involved in stabilization of the cationic sugar and the anionic uracil leaving group, respectively, specifically
weakens binding of I to the UDG-uracil complex by 154,000-fold, without significantly affecting substrate or product binding.
These results suggest that electrostatic effects can effectively stabilize such an intermediate by at least â7 kcal/mol, without
leading to anticatalytic stabilization of the substrate and products.
The Escherichia coli enzyme 3-methyladenine DNA glycosylase I (TAG) hydrolyzes the glycosidic bond of 3-methyladenine (3-MeA) in DNA and is found in many bacteria and some higher eukaryotes. TAG ...shows little primary sequence identity with members of the well-known helix-hairpin-helix (HhH) superfamily of DNA repair glycosylases, which consists of AlkA, EndoIII, MutY and hOGG1. Unexpectedly, the three-dimensional solution structure reported here reveals TAG as member of this superfamily. The restricted specificity of TAG for 3-MeA bases probably arises from its unique aromatic rich 3-MeA binding pocket and the absence of a catalytic aspartate that is present in all other HhH family members.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
DNA glycosylases initiate base excision repair by removing damaged or mismatched bases, producing apurinic/apyrimidinic (AP) DNA. For many glycosylases, the AP-DNA remains tightly bound, impeding ...enzymatic turnover. A prominent example is thymine DNA glycosylase (TDG), which removes T from G·T mispairs and recognizes other lesions, with specificity for damage at CpG dinucleotides. TDG turnover is very slow; its activity appears to reach a plateau as the product/enzyme ratio approaches unity. The follow-on base excision repair enzyme, AP endonuclease 1 (APE1), stimulates the turnover of TDG and other glycosylases, involving a mechanism that remains largely unknown. We examined the catalytic activity of human TDG (hTDG), alone and with human APE1 (hAPE1), using pre-steady-state kinetics and a coupled-enzyme (hTDG-hAPE1) fluorescence assay. hTDG turnover is exceedingly slow for G·T (kcat = 0.00034 min-1) and G·U (kcat = 0.005 min-1) substrates, much slower than kmax from single turnover experiments, confirming that AP-DNA release is rate-limiting. We find unexpectedly large differences in kcat for G·T, G·U, and G·FU substrates, indicating the excised base remains trapped in the product complex by AP-DNA. hAPE1 increases hTDG turnover by 42- and 26-fold for G·T and G·U substrates, the first quantitative measure of the effect of hAPE1. hAPE1 stimulates hTDG by disrupting the product complex rather than merely depleting (endonucleolytically) the AP-DNA. The enhancement is greater for hTDG catalytic core (residues 111–308 of 410), indicating the N- and C-terminal domains are dispensable for stimulatory interactions with hAPE1. Potential mechanisms for hAPE1 disruption of the of hTDG product complex are discussed.
Abstract only
The DNA base excision repair (BER) pathway is essential for maintaining genomic integrity and is implicated in active DNA demethylation, a key element of epigenetic transcriptional ...regulation. Thymine DNA glycosylase (TDG) excises thymine from mutagenic G·T mispairs, initiating repair of deaminated 5‐methylcytosine (mC). TDG also excises 5‐formylcytosine (fC) and 5‐carboxylcytosine (caC), oxidation products of mC produced by Tet enzymes. These seemingly disparate activities are consistent with TDG specificity for acting at CpG sites and its essential role in active DNA demethylation and embryonic development. Understanding how glycosylases excise lesions and avoid acting on undamaged DNA is an important problem in DNA repair. Structural and biochemical results here reveal how TDG attains broad specificity for G·T and G·fC lesions while avoiding A·T pairs. A crystal structure of TDG (catalytic domain) bound to substrate analogue suggests G·T glycosylase activity is suboptimal owing to unfavorable interactions between flipped dT substrate and two active‐site residues. Remarkably, mutating these residues greatly increases G·T activity and confers substantial activity for normal A·T base pairs. The results suggest TDG evolved with suboptimal G·T repair capability in order to minimize aberrant activity on undamaged DNA, an unprecedented finding for a repair enzyme. Supported by NIH (R01‐GM072711).
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