Ionic strength and magnesium affect the specificity of Escherichia coli and human 8-oxoguanine-DNA glycosylases Viktoriya S Sidorenko1, Grigory V Mechetin1, Georgy A Nevinsky1,2 and Dmitry O Zharkov1,2 SB RAS Institute of Chemical Biology and Fundamental Medicine, Novosibirsk, Russia Department of Natural Sciences, Novosibirsk State University, Russia Keywords 8-oxoguanine; DNA damage; DNA glycosylase; DNA repair; substrate specificity Correspondence D O Zharkov, SB RAS Institute of Chemical Biology and Fundamental Medicine, Novosibirsk 630090, Russia Fax: +7 383 333 3677 Tel: +7 383 335 6226 E-mail: dzharkov@niboch.nsc.ru (Received 20 February 2008, revised 18 April 2008, accepted 23 May 2008) doi:10.1111/j.1742-4658.2008.06521.x An abundant oxidative lesion, 8-oxo-7,8-dihydroguanine (8-oxoG), often directs the misincorporation of dAMP during replication To prevent mutations, cells possess an enzymatic system for the removal of 8-oxoG A key element of this system is 8-oxoguanine-DNA glycosylase (Fpg in bacteria, OGG1 in eukaryotes), which must excise 8-oxoG from 8-oxoG:C pairs but not from 8-oxoG:A We investigated the influence of various factors, including ionic strength, the presence of Mg2+ and organic anions, polyamides, crowding agents and two small heterocyclic compounds (biotin and caffeine) on the activity and opposite-base specificity of Escherichia coli Fpg and human OGG1 The activity of both enzymes towards 8-oxoG:A decreased sharply with increasing salt and Mg2+ concentration, whereas the activity on 8-oxoG:C was much more stable, resulting in higher opposite-base specificity when salt and Mg2+ were at near-physiological concentrations This tendency was observed with both Cl) and glutamate as the major anions in the reaction mixture Kinetic and binding parameters for the processing of 8-oxoG:C and 8-oxoG:A by Fpg and OGG1 were determined under several different conditions Polyamines, crowding agents, biotin and caffeine affected the activity and specificity of Fpg or OGG1 only marginally We conclude that, in the intracellular environment, the specificity of Fpg and OGG1 for 8-oxoG:C versus 8-oxoG:A is mostly due to high ionic strength and Mg2+ In all living organisms DNA is subject to ongoing damage by various environmental and endogenous factors [1] One of the most frequently encountered base lesions is 8-oxo-7,8-dihydroguanine (8-oxoG), produced by oxidative stress to the steady-state level of $ · 106 guanines in human DNA [2] 8-oxoG is mutagenic due to its ability to form a stable Hoogsten pair with A [3] and its propensity to direct the incorporation of dAMP by DNA polymerases [4] If left uncorrected, the resulting 8-oxoG:A mispair is converted to a T:A pair in the next round of replica- tion, producing a G:C fi T:A transversion mutation, the type frequently encountered in human cancers [5,6] The consequences of 8-oxoG’s appearance in DNA are counteracted by a three-tier enzymatic ‘GO system’ [7–9], part of general base-excision repair system [10] In bacteria, once it has emerged in DNA in the context of a G:C pair, the 8-oxoG base is excised from the 8-oxoG:C pair by formamidopyrimidine-DNA glycosylase (Fpg, EC 3.2.2.23); in eukaryotes it is excised by 8-oxoguanine-DNA glycosylase OGG1, followed by Abbreviations 8-oxoG, 8-oxo-7,8-dihydroguanine; AP, apurinic ⁄ apyrimidinic; KGlu, potassium glutamate; THF, tetrahydrofuran FEBS Journal 275 (2008) 3747–3760 ª 2008 The Authors Journal compilation ª 2008 FEBS 3747 Factors affecting the specificity of Fpg and OGG1 V S Sidorenko et al repair to restore the original G:C pair Importantly, both Fpg and OGG1 are much less likely to excise 8-oxoG from 8-oxoG:A substrates, because if these mispairs are generated by the incorporation of dAMP opposite 8-oxoG, such excision would immediately fix the G:C fi T:A transversion Instead, 8-oxoG:A mispairs are processed by removal of A by the DNA glycosylases MutY (in bacteria) or MUTYH (in eukaryotes) and conversion of 8-oxoG:A to 8-oxoG:C in the first round of repair, followed by a Fpg- or OGG1-initiated second round of repair The third member of the GO system, MutT ⁄ NUDT1 protein, hydrolyzes 8-oxodGTP, thus preventing incorporation of 8-oxoG into DNA during replication [7–9] Inactivation of the GO system increases the mutagenesis rate in bacteria [11] and increases the risk of cancer development in mouse models [12,13] and in humans [14,15] Discrimination in favor of 8-oxoG:C and against 8-oxoG:A mispairs by Fpg and OGG1 is a key feature on which the GO system is built Although Fpg and OGG1 share no similarity in either their sequence or structure, the crystal structures of these proteins reveal extensive sets of bonds with the C base opposite the lesion [16–18] Furthermore, stopped-flow studies suggest that additional discrimination of the base opposite the lesion may occur at earlier stages of substrate binding by both enzymes [19,20] Although published studies on the opposite-base specificity of Fpg and OGG1 [19–26] agree that 8-oxoG:C substrates are preferred over 8-oxoG:A substrates, the multitude of used assay systems precludes a systematic analysis of the influence that may be exerted by various reaction factors on this specificity In most kinetic studies, the activity of DNA glycosylases is assayed in well-defined systems that include a buffer (often non-physiological, such as Tris or Good buffers), a salt (usually NaCl or KCl) and stabilizing agents (usually a metal chelator, a thiol reagent and glycerol) In living cells, the reactions catalyzed by DNA-dependent proteins may be affected by ionic strength, the concentration of divalent cations such as Mg2+, the nature of the buffering agents, the presence of competing polyamines and other small molecules, and crowding by other macromolecules Their effects on the specificity of 8-oxoG excision have never been studied Because these factors may be important for the efficiency of correct 8-oxoG repair, in this study we address how the relative efficiency of 8-oxoG excision from pairs with C and A by Fpg and OGG1 depends on buffer composition, ionic strength, Mg2+ concentration and several other factors 3748 Results Effects of ionic strength and divalent cations on the activity and specificity of Fpg and OGG1 The conditions inside a living cell differ from most buffer systems in which the activity and specificity of Fpg and OGG1 have been studied For example, in Escherichia coli the intracellular concentrations of Na+ and Cl) ions are $ mm, the major intracellular monovalent cation is K+ ($ 200–250 mm), free divalent cations are mostly Mg2+ ($ 10 mm) and anions are represented by a mixture of organic acids, amino acids, inorganic phosphate and nucleic acids [27] To explore the dependence of the activity and specificity of Fpg and OGG1 on general ionic strength and the presence of divalent cations, we conducted a factorial design experiment in which both factors were varied by changing the concentration of KCl and MgCl2, whereas the buffering agent (potassium phosphate, KPi) was kept constant at 25 mm (Table 1; Series 1) Processing of 8-oxoG:C and 8-oxoG:A substrates was followed in a single time point assay in a linear kinetics range Mechanistically, Fpg and OGG1 differ in their ability to catalyze cleavage of the apurinic ⁄ apyrimidinic (AP) site via elimination of its 3¢-phosphate (AP lyase activity) after excision of the damaged base (DNA glycosylase activity) Fpg efficiently catalyzes b,d-elimination at the AP site so that these reactions cannot be separated kinetically [23,28]; therefore, unassisted cleavage of substrate DNA by this enzyme was used as the assay endpoint The AP lyase activity of OGG1 proceeds via b-elimination and is much less efficient than its glycosylase activity [25,26] Two assay endpoints were used in this case: glycosylase activity was measured after full thermal degradation of the AP site left by base excision, whereas the AP lyase activity was assayed as unassisted cleavage of the substrate by Table Outline of the factorial design activity experiments Series Varied reaction mixture components Concentrations KCl MgCl2 KGlu MgCl2 KPi Spermine or spermidine MgCl2 Poly(ethylene glycol) 4000 or 8000 MgCl2 0, 50, 100, 150, 200 mM 0, 5, 10, 15, 20 mM 0, 50, 100, 150, 200 mM 0, 5, 10, 15, 20 mM 0, 25 mM 0, 1, 10, 100, 1000 lM 0, 5, 10, 15, 20 mM 0, 0.05, 0.1, 0.2, 0.5, 1, 2, 5% 0, 5, 10, 15, 20 mM FEBS Journal 275 (2008) 3747–3760 ª 2008 The Authors Journal compilation ª 2008 FEBS V S Sidorenko et al Factors affecting the specificity of Fpg and OGG1 the enzyme In the experiments described here, all these activities were assayed unless indicated otherwise Both enzymes showed a decrease in the efficiency of cleavage of both substrates with increasing ionic strength and Mg2+ concentration (Fig 1) However, in all cases, the activities of both Fpg and OGG1 on 8-oxoG:A decreased much more sharply than on the 8-oxoG:C Whereas in the absence of KCl and MgCl2 in the reaction mixture, the activities on these substrates were at least of the same order of magnitude and the opposite-base specificity (C ⁄ A specificity, A defined as the ratio of cleavage of the 8-oxoG:C to 8-oxoG:A substrate under identical conditions) in all cases was the lowest, an increase in both salts to nearphysiological concentrations led to an $ 10–50-fold preference for C compared with A opposite the lesion The activity of Fpg on 8-oxoG:C was more sensitive to variations in buffer composition than the activity of OGG1 on the same substrate (compare Fig 1A,D and G); OGG1 was inhibited only by the highest concentrations of KCl and MgCl2 Interestingly, the AP lyase activity of OGG1 on 8-oxoG:A at any given B 50 60 50 40 30 20 10 20 10 00 Mg Cl 10 , m 15 M 50 10 mM 15 100 20 150 200 K ,m Cl M D C/A 30 Mg Cl 2, 12 10 00 [P], nM [P], nM 40 C 100 20 150 200 K ,m Cl M E 10 00 Mg Cl 10 2, m M Mg Cl 2, 100 15 20 150 200 l, m KC M G 00 50 mM 15 Mg Cl 10 , m 15 M 50 10 100 20 150 200 KC l, m M 00 Mg Cl 10 2, m M 50 100 15 20 l, m KC 150 200 M l KC ,m M 50 100 20 150 200 K ,m Cl M 12 10 00 Mg Cl 10 , m 15 M 12 10 00 C/A 200 I [P], nM [P], nM 150 35 30 25 20 15 10 00 H 10 100 20 C/A [P], nM 15 [P], nM 20 30 10 50 F 40 20 Mg Cl 10 , m 15 M 50 50 100 20 150 200 l KC ,m M Mg Cl 10 2, m M 50 100 15 20 150 200 l KC ,m M Fig Activity and specificity of Fpg and OGG1 in buffers with different concentrations of KCl and MgCl2 (A–C) Fpg, (D–F) glycosylase activity of OGG1, (G–I) AP lyase activity of OGG1 The extent of cleavage of 8-oxoG:C (A, D, G) or 8-oxoG:A (B, E, H) or the C ⁄ A specificity (C, F, I) is plotted against the concentrations of the salts [P], product concentration Note the different scales in (A, D, G) compared with (B, E, F) The concentration of the enzyme and both substrates were kept constant in the analysis of each activity (see Experimental procedures for the reaction conditions) Means of two independent experiments are shown FEBS Journal 275 (2008) 3747–3760 ª 2008 The Authors Journal compilation ª 2008 FEBS 3749 Factors affecting the specificity of Fpg and OGG1 V S Sidorenko et al concentration of KCl decreased more slowly than its DNA glycosylase activity Overall, the highest opposite-base specificity for Fpg was observed at 5–15 mm MgCl2 and 50–150 mm KCl (Fig 1C), for OGG1 glycosylase activity, at 5–10 mm MgCl2 and 150– 200 mm KCl (Fig 1F) and for its AP lyase activity, at 10–20 mm MgCl2 and 100–200 mm KCl (Fig 1I) To analyze the impact of Mg2+ on the oppositebase specificity of Fpg in more detail, we measured the steady-state kinetic parameters for the cleavage of 8-oxoG:C and 8-oxoG:A by this enzyme in the presence and absence of 10 mm MgCl2 The concentration of KCl in these experiments was 50 mm, because higher ionic strengths improved Fpg specificity at the cost of a reduction in 8-oxoG:A cleavage to very low levels, making the determination of individual kinetic constants problematic The C ⁄ A preference measured in the factorial design experiments under these conditions was 5.2 for mm MgCl2 and 16 for 10 mm MgCl2 The results are summarized in Table In the absence of Mg2+, the kinetic constants were in agreement with those reported in the literature [23], with 8-oxoG:C being a better substrate because of its lower KM value The effect of Mg2+ on KM was not high ($ 1.5-fold); however, in the presence of Mg2+, KM improved for 8-oxoG:C and worsened for 8-oxoG:A By contrast, Mg2+ reduced kcat in both cases, possibly due to the induction of conformational changes in the DNA molecule that interfere with those required for catalysis by Fpg [29] Thus, KM values better reflected the changes in Fpg opposite-base specificity induced by Mg2+ in single time point factorial design experiments OGG1 generally does not display Michaelis–Menten kinetics due to the slow release of the reaction product [30] However, it is possible to describe the action of this enzyme by a three-step kinetic scheme (Scheme 1) and determine two individual rate constants, k2 and k3, which describe the processes of base excision and product release, respectively, using single turnover kinetics for k2 and burst rate kinetics for k3 [30,31] Table Kinetic constants of cleavage of 8-oxoG:C and 8-oxoG:A substrates by Fpg in the presence and in the absence of Mg2+ Mean ± SD of three independent experiments Substrate 8-oxoG:C 8-oxoG:A 3750 MgCl2 (mM) KM (nM) kcat (min)1) kcat ⁄ KM (nM)1Ỉmin)1) 10 10 26 17 490 700 6.1 1.2 3.3 1.0 0.24 0.068 0.0066 0.0015 ± ± ± ± 150 340 ± ± ± ± 1.2 0.2 0.7 0.4 k1 k2 k3 E ỵ S  ES ! EP ! E ỵ P k1 Scheme To independently evaluate the effects of ionic strength and Mg2+ on the activity and specificity of OGG1, we measured the apparent values of k2 and k3 under conditions of low salt (KPi only) and no Mg2+, low salt and 20 mm Mg2+, and high salt (KPi + 150 mm KCl) and no Mg2+ These conditions were selected to represent regions of the factorial design experiments markedly different in OGG1 specificity (Fig 1F), i.e low preference for 8-oxoG:C versus 8-oxoG:A in low salt and no Mg2+ (C ⁄ A specificity of 1.5 for DNA glycosylase reaction and 0.78 for AP lyase reaction) and the increase in the preference for 8-oxoG:C with increasing salt (C ⁄ A specificity of 8.6 for DNA glycosylase reaction and 1.7 for AP lyase reaction in 150 mm KCl, mm MgCl2) or Mg2+ (C ⁄ A specificity of 18 for DNA glycosylase reaction and 6.7 for AP lyase reaction in mm KCl, 20 mm MgCl2) The results are summarized in Table The k2 and k3 constants did not show much variation for 8-oxoG:C over the set of conditions tested, with a maximum 2.5-fold difference in k2 and a 2.2-fold difference in k3, and both rate constants improved on addition of MgCl2 or KCl However, an increase in the ionic strength of Mg2+ had a pronounced deleterious effect on k2 and k3 for 8-oxoG:A substrates: 20 mm Mg2+ decreased k2 by 42-fold and k3 by 5.5-fold, whereas 150 mm KCl decreased k2 by 3.9-fold and k3 by 6.9fold Therefore, physiological concentrations of ionic strength and divalent cations enhance both base excision and the turnover of OGG1 cleaving its proper substrate, 8-oxoG:C, and prevent cleavage of the improper 8-oxoG:A substrate A well-recognized mechanism by which ionic strength and divalent cations could modulate the activity of DNA-dependent enzymes is changes in the affinity of the enzymes for their DNA substrates For example, binding of Fpg to damaged DNA shows a bell-shaped dependence with a peak at $ 100 mm KCl and an approximately twofold decrease in binding at and 500 mm KCl [32] Thus, to address the influence of the reaction conditions on the binding of Fpg and OGG1 to damaged DNA, we determined Kd values for binding under the same conditions as used for the kinetic experiments In these experiments, fluorescence titration was the method of choice because it allows full control over the composition of the reaction mixture To minimize the impact of protein binding to non-damaged DNA, shortened 12-mer ligands were used, identical in sequence to the central part of the 23-mers used in the kinetic experiments but containing an uncleavable FEBS Journal 275 (2008) 3747–3760 ª 2008 The Authors Journal compilation ª 2008 FEBS V S Sidorenko et al Factors affecting the specificity of Fpg and OGG1 Table Rate constants of cleavage of 8-oxoG:C and 8-oxoG:A substrates by OGG1 under different conditions Mean ± SD of 3–5 independent experiments 8-oxoG:C 8-oxoG:A )1 )1 k2 (min)1) k3 (min)1) Conditions k2 (min ) k3 (min ) mM KCl mM MgCl2 mM KGlu 0.50 ± 0.09 0.21 ± 0.11 0.97 ± 0.49 0.011 ± 0.008 mM KCl 20 mM MgCl2 mM KGlu 1.1 ± 0.4 0.22 ± 0.03 0.023 ± 0.008 0.0020 ± 0.0013 150 mM KCl mM MgCl2 mM KGlu 1.3 ± 0.6 0.47 ± 0.06 0.25 ± 0.10 0.0016 ± 0.0004 mM KCl mM MgCl2 200 mM KGlu 0.58 ± 0.16 0.14 ± 0.02 1.66 ± 0.68 0.0007 ± 0.001a a Large error is due to fitting to a very shallow-slope linear curve tetrahydrofuran (THF) moiety instead of 8-oxoG THF is a good ligand for Fpg and OGG1, with their affinity for THF-containing DNA closely paralleling the affinity for 8-oxoG-containing DNA [23,26], and these particular ligands have been successfully used to analyze stopped-flow kinetics for both enzymes [19,20] The results of the fluorescence titration experiments are summarized in Fig In the absence of MgCl2, the affinity of Fpg for the THF:C ligand was 1.6-fold higher than for the THF:A ligand The presence of Mg2+ had little effect on the binding of Fpg to the THF:C ligand and slightly improved binding to the THF:A ligand, making it comparable with binding to THF:C (Fig 2B, groups and 2) Therefore, it is unlikely that the 2.0 B 1.5 Kd, µM Fluorescence, a.u A observed decrease in enzyme activity on 8-oxoG:A and the concomitant increase in C ⁄ A specificity are due to differences in binding In the case of OGG1, the affinity of the enzyme for THF:C was 3.5-fold higher than for THF:A in the absence of MgCl2 and at low ionic strength Addition of 150 mm KCl did not change the situation much, whereas addition of 20 mm MgCl2 increased the Kd values for both ligands, with THF:C affected more than THF:A but still preferred by OGG1 (Fig 2B, groups 3–5) As with Fpg, these observations not support the idea that binding of the glycosylase to damaged DNA contributes significantly to ionic strength and the effects of Mg2+ on enzyme specificity 1.0 0.5 0.0 [ligand], µM CA CA CA CA CA CA Fig Binding of Fpg and OGG1 to uncleavable THF:C and THF:A ligands under different conditions (A) A representative experiment showing fluorescence titration of Fpg with a THF:C ligand in the presence of mM (black circles) or 10 mM (white circles) MgCl2 AU, arbitrary units (B) Dissociation constants for binding of Fpg (1, 2) and OGG1 (3–6) to THF:C and THF:A ligands (denoted C and A, and represented by white and black circles, respectively) determined from the fluorescence titration data The variable components of the buffers included: 25 mM KPi and 50 mM KCl (1), 25 mM KPi, 50 mM KCl and 10 mM MgCl2 (2), 25 mM KPi (3), 25 mM KPi and 20 mM MgCl2 (4), 25 mM KPi and 150 mM KCl (5), 25 mM KPi and 200 mM KGlu (6) (see also Tables and 3) The mean ± SD of two independent experiments is shown FEBS Journal 275 (2008) 3747–3760 ª 2008 The Authors Journal compilation ª 2008 FEBS 3751 Factors affecting the specificity of Fpg and OGG1 V S Sidorenko et al Effects of organic anions on the specificity of Fpg and OGG1 It has been reported previously that the activity and specificity of some enzymes can depend on the presence of organic anions in the reaction For example, a typical organic anion, glutamate, has been found to improve the efficiency of DNA synthesis by DNA polymerase I or its Klenow fragment, as well as their ability to bypass DNA lesions, in comparison with Cl) [33] Because organic anions represent a major fraction of total ions and buffering species in the cell, we investigated how the presence of glutamate affects the activity and specificity of Fpg and OGG1 Two factorial design experiments were performed, one with potassium glutamate (KGlu) replacing KCl as a salt in the presence of KPi as the main buffering agent, another with KGlu as the sole salt and buffer; the Mg2+ concentration was varied in the same way as in the KPi– KCl experiments described above (Table 1, Series 2) For Fpg, the substitution of KGlu for KCl did not change the overall dependence of the enzyme activity if KPi was present (Fig 3A–C) The only notable difference was a higher activity towards 8-oxoG:C at high Mg2+ and salt concentrations compared with when Cl) was the major anion (cf Figs 1A and 3A) As a consequence, the specificity of Fpg for 8-oxoG:C versus 8-oxoG:A was highest at 150–200 mm KGlu and 5– 20 mm MgCl2, conditions that may better resemble the cellular environment If KPi was absent (Fig 4A–C), Fpg had very low activity at mm KGlu and mm MgCl2, possibly because no ionic strength was provided (except 1.25 mm KPi from the enzyme dilution buffer) However, KGlu as a sole buffer supported a substantially high activity of Fpg towards 8-oxoG:C at all other concentrations of KGlu and MgCl2, and towards 8-oxoG:A at 0–5 mm MgCl2 Overall, the C ⁄ A specificity in this case also increased with increasing salt and MgCl2 In the case of OGG1, replacing KCl with KGlu did not have much influence on enzyme glycosylase activity towards 8-oxoG:C (cf Figs 1D and 3D) The glycosylase activity of OGG1 towards 8-oxoG:A was highest at mm KGlu and mm MgCl2 and decreased at higher concentrations, but, unlike the situation observed with KCl, it remained essentially unchanged as the salt concentrations increased (Fig 3E) As a result, the C ⁄ A specificity of this reaction was highest at low to medium concentrations of KGlu (0–50 mm) and MgCl2 (0–10 mm), whereas the activity towards 8-oxoG:C was higher The AP lyase reaction with 8-oxoG:A was efficient only at low Mg2+ and no KGlu, whereas with 8-oxoG:C it was in general 3752 agreement with the salt dependence of the glycosylase reaction; the C ⁄ A specificity was highest at low to medium KGlu and medium MgCl2 In the absence of KPi, the DNA glycosylase and AP lyase activity of OGG1 towards 8-oxoG:C resembled its activity in the presence of KPi (Fig 4G,H) However, exclusion of KPi significantly influenced both activities of OGG1 with 8-oxoG:A; a more or less efficient glycosylase reaction was observed only at mm KGlu and 0– 15 mm MgCl2, whereas the AP lyase reaction required 0–100 mm KGlu and 0–5 mm MgCl2 The C ⁄ A specificity of the glycosylase reaction in the absence of KPi was usually higher than in the presence of KPi due to less efficient cleavage of 8-oxoG:A; the highest specificity was observed at mm KGlu + 20 mm MgCl2 and 200 mm KGlu + mm MgCl2, where cleavage of 8-oxoG:A was minimal The overall C ⁄ A specificity of the AP lyase reaction was highest at high KGlu and low to intermediate MgCl2 concentrations Interestingly, OGG1, unlike Fpg, displayed robust activity on both substrates in the absence of KPi, KGlu and MgCl2 To dissect the kinetic contribution of a high KGlu concentration to the specificity of OGG1, we also determined the values k2 and k3 with both 8-oxoG:C and 8-oxoG:A in the presence of 25 mm KPi and 200 mm KGlu (C ⁄ A specificity 9.0 for the glycosylase reaction, 15 for the AP lyase reaction) As shown in Table 3, in comparison with KPi only, the addition of KGlu had a minimal effect on either rate constant in the case of 8-oxoG:C (a 1.2-fold increase in k2 and an 1.5-fold decrease in k3), and even improved the k2 value for 8-oxoG:A by 1.7-fold However, this was accompanied by a 16-fold decrease in k3, indicating that the enzyme turnover on 8-oxoG:A slows significantly, contributing to a decrease in the efficiency of its cleavage by OGG1 Moreover, fluorescence titration analysis of OGG1 binding to uncleavable THF:C and THF:A damaged ligands showed that although KGlu did not affect the affinity of the enzyme for the THF:C ligand, its affinity for the THF:A ligand decreased at least 2.3-fold in comparison with the reactions (C ⁄ A specificity for binding was 3.5 for 25 mm KPi, 2.8 for 25 mm KPi + 150 mm KCl, and 8.6 for 25 mm KPi + 200 mm KGlu) Thus, the presence of KGlu may also disfavor the 8-oxoG:A substrate at the level of binding Polyamines, crowding agents and some purine analogs not affect the activity and specificity of 8-oxoguanine-DNA glycosylases Several factors that may, in principle, affect the efficiency of 8-oxoG excision by Fpg and OGG1 have FEBS Journal 275 (2008) 3747–3760 ª 2008 The Authors Journal compilation ª 2008 FEBS V S Sidorenko et al Factors affecting the specificity of Fpg and OGG1 A B C 40 50 15 30 C/A 30 [P], nM [P], nM 40 10 20 10 00 Mg Cl 10 , m 15 M Mg Cl 10 2, m 50 100 150 200 K u, Gl 10 00 20 mM M 50 100 15 20 150 200 K u, Gl mM M 20 Mg 10 Cl , m 15 M 12 10 00 20 150 200 l KG m u, M G 20 m u, Gl K M 150 200 00 Mg Cl 10 2, m 50 100 20 150 200 K u, Gl mM H 30 25 20 15 10 00 100 15 10 Mg Cl 10 , m 15 M 50 100 50 F C/A 30 M 50 100 15 20 150 200 K u, Gl mM I 15 [P], nM [P], nM Mg Cl 50 10 ,m 15 M 20 100 150 200 K m u, Gl M C/A 10 Mg Cl 10 2, m 50 [P], nM [P], nM 40 00 E D 20 10 00 Mg C 50 10 l2 , 15 mM 100 20 m u, Gl K 150 200 M Mg Cl 10 , m 15 M 50 100 20 150 200 K u, Gl m M Fig Activity and specificity of Fpg and OGG1 in buffers with different concentrations of KGlu and MgCl2 in the presence of KPi (A–C) Fpg, (D–F) glycosylase activity of OGG1, (G–I) AP lyase activity of OGG1 The extent of cleavage of 8-oxoG:C (A, D, G) or 8-oxoG:A (B, E, H) or the C ⁄ A specificity (C, F, I) is plotted against the concentrations of the salts [P], product concentration Note the different scales in panels (A, D, G) compared with (B, E, F) The means of two independent experiments are shown never been investigated Nucleic acids in bacteria and human cells are bound to polyamines (spermine, spermidine and putrescine), abundant products of amino acid metabolism with important structural and regulatory functions [34] Because polyamine binding affects the structure of nucleic acids and the availability of their hydrogen-bond donors and acceptors, the activities of DNA-dependent enzymes may be influenced by polyamine binding to their DNA substrates; for example, polyamines activate poly(ADP-ribose) polymerase [35] and improve the fidelity of HIV-1 reverse transcriptase [36] We investigated the cleavage of 8-oxoG:C and 8-oxoG:A substrates by Fpg (not shown) and OGG1 (Fig 5A,B) in the presence of 0–1000 lm spermine or spermidine (Table 1, Series 3) and varying concentrations of MgCl2 In the absence of Mg2+, spermine slightly ($ 1.5-fold) increased the specificity of Fpg due to a corresponding decrease in activity on 8-oxoG:A No significant influence of polyamines on OGG1 activity was observed, except that AP lyase activity on 8-oxoG:C was approximately twofold higher in mm spermine (but not spermidine), possibly due to the chemical degradation of AP sites by polyamines [37] Overall, polyamines had minimal FEBS Journal 275 (2008) 3747–3760 ª 2008 The Authors Journal compilation ª 2008 FEBS 3753 Factors affecting the specificity of Fpg and OGG1 A V S Sidorenko et al B 50 30 25 20 15 10 00 40 Mg Cl 10 , m 15 M 20 150 200 l KG u, m M D 30 Mg Cl 10 , m 15 M 50 100 C/A [P], nM [P], nM 60 50 40 30 20 10 00 C 10 00 Mg Cl 10 , m 15 M 50 100 20 150 200 K u, Gl mM E 35 30 25 20 15 10 10 00 Mg 10 Cl , m 15 M 20 150 200 K u, Gl m M G Mg C 50 100 00 10 l2 , mM15 100 20 150 200 l KG u, mM 10 00 Mg Cl 50 10 ,m 15 M 20 100 150 200 l KG m u, M M 50 100 20 150 200 l KG u, mM 14 12 10 00 60 50 40 30 20 10 00 C/A 20 200 m I [P], nM [P], nM 30 lu, KG 150 Mg Cl 10 , m 15 M 50 H 40 20 C/A [P], nM 15 [P], nM 20 40 20 10 50 100 F 50 30 20 Mg Cl 10 , m 15 M 50 100 20 m lu, KG 150 200 M Mg Cl 10 , m 15 M 50 100 20 150 200 l KG u, m M Fig Activity and specificity of Fpg and OGG1 in buffers with different concentrations of KGlu and MgCl2 in the absence of KPi (A–C) Fpg, (D–F) glycosylase activity of OGG1, (G–I) AP lyase activity of OGG1 The extent of cleavage of 8-oxoG:C (A, D, G) or 8-oxoG:A (B, E, H) or the C ⁄ A specificity (C, F, I) is plotted against the concentrations of the salts [P], product concentration Note the different scales in (A, D, G) compared with (B, E, F) Means of two independent experiments are shown influence on the activity and specificity of both Fpg and OGG1 Another factor that can seriously influence the activities of various enzymes in the cell is its crowding with macromolecular agents [38], and crowding has been shown to modulate DNA-dependent enzymes such as DNA ligases [39] or restriction endonucleases [40] Poly(ethylene glycol) fractions of differing average molecular masses are widely used as crowding agents in enzyme kinetics We investigated the activity of Fpg and OGG1 towards 8-oxoG:C and 8-oxoG:A in the presence of 0–5% poly(ethylene glycol) 4000 or 8000 3754 and varying concentrations of MgCl2 (Table 1, Series 4) and found only marginal differences for any of the enzyme–substrate pairs [Fig 5C shows an example of DNA glycosylase activity of OGG1 with the range of poly(ethylene glycol) 8000 and mm MgCl2] Therefore, macromolecular crowding is likely to be of little importance for the function of these two enzymes In addition, we analyzed the effect of two low molecular mass compounds, biotin and caffeine, on the activity of Fpg and OGG1 Biotin can be regarded as a structural mimic of 8-oxopurines [41], and avidin, FEBS Journal 275 (2008) 3747–3760 ª 2008 The Authors Journal compilation ª 2008 FEBS V S Sidorenko et al Factors affecting the specificity of Fpg and OGG1 200 A B C % Activity 150 100 50 10 1 10 00 10 10 10 00 10 10 0 PEG 8000, % Spermine, µM Fig Activity of OGG1 in the presence of polyamines and crowding agents (A) DNA glycosylase and (B) AP lyase activity on the 8-oxoG:C substrate in the presence of spermine (0 mM MgCl2) (C) DNA glycosylase activity on the 8-oxoG:C substrate in the presence of poly(ethylene glycol) 8000 (0 mM MgCl2) The activity in the presence of spermine or poly(ethylene glycol) is normalized to the same activity in their absence (100%); the scale is the same in all panels Means ± SD of three independent experiments are shown; in some cases, the error bars are hidden by the symbols a well-known biotin-binding protein, has been shown by X-ray crystallography to bind 8-oxopurines in its biotin-binding site [42]; thus, the possibility of biotin association with 8-oxoG-binding sites of DNA glycosylases could not be excluded Caffeine, the most widely ingested xenobiotic purine base in the world, apparently influences several pathways of DNA repair through mechanisms that are not fully understood [43] We analyzed the ability of Fpg and OGG1 to cleave their substrates in the presence of up to 20 mm biotin or caffeine However, no effect was found except for a slight ($ 30%) inhibition of Fpg at the highest caffeine concentration used (data not shown) Therefore, biotin and caffeine are unlikely to influence the activities of these enzymes in vivo Discussion The substrate specificity of DNA glycosylases has been subject to a number of studies, yet the results are often conflicting For example, Fpg has been reported to excise more than 20 different damaged bases from oligonucleotide substrates [44], whereas the excision from damaged genomic DNA has been reported only for 8-oxoG, 4,6-diamino-5-formamidopyrimidine, 2,4-diamino-6-oxo-5-formamidopyrimidine and 2,4-diamino6-oxo-5N-methyl-5-formamidopyrimidine [45,46] The relative activity of Fpg on substrates containing different bases opposite 8-oxoG also seemingly varies depending on the assay used [19,23] It is clear that when an enzyme can process several substrates with comparable efficiencies, as is the case for almost all DNA glycosylases [47], the preferences for each substrate may depend on the reaction conditions to different degrees The influence of the reaction conditions on various aspects of substrate specificity of DNA glycosylases has been given little attention, but there are reasons to believe that the impact of ionic strength and divalent cations may be significant In one recent study, submillimolar concentrations of Mg2+ have been shown to stimulate the excision of hypoxanthine but not of 1,N6-ethenoadenine by murine methylpurine-DNA glycosylase (MPG) [48] Regarding the opposite-base specificity, discrimination of the opposite base by human endonuclease III is strongly dependent on Mg2+ concentrations, approaching its maximum at 10–20 mm MgCl2 [49] Our study explicitly addressed the opposite-base specificity of two 8-oxoguanine-DNA glycosylases, the only DNA glycosylases for which the preference for a particular base opposite the lesion has been proved to play a biologically important role [7–9] Both Fpg and OGG1 display a strong preference for 8-oxoG:C in comparison with 8-oxoG:A [21,23,26,50] We were interested in a systematic analysis of this opposite-base preference, and, in particular, how it may change under conditions approximating the intracellular environment Two principal factors that affected the opposite-base specificity of Fpg and OGG1 were general ionic strength and Mg2+ concentration In general, the specificity of both enzymes was highest when these factors approached physiological values The reason for the increase in specificity was a pronounced decrease in the activity of Fpg and OGG1 on 8-oxoG:A at high Mg2+ and ionic strength, whereas most of the activity on 8-oxoG:C was retained under these conditions At least for OGG1, we observed only a modest decrease in the affinity for both THF:C and THF:A uncleavable ligands with increasing salt concentration In the case of Fpg, it has been reported that binding FEBS Journal 275 (2008) 3747–3760 ª 2008 The Authors Journal compilation ª 2008 FEBS 3755 Factors affecting the specificity of Fpg and OGG1 V S Sidorenko et al of this enzyme to uncleavable damaged DNA ligands is also moderately affected by salt concentration (approximately twofold difference between the best and the worst binding in the 0–500 mm KCl range) [32] Therefore, general affinity does not seem to contribute much to the effect of ionic strength on glycosylase activity and specificity However, ionic strength may possibly have a selective effect on some stages of multistage lesion recognition by Fpg and OGG1 [19,20] Although no structural information on Fpg or OGG1 complexed with DNA containing A opposite the lesion is available, the structures of both enzymes complexed with undamaged DNA show that initial recognition of the lesion involves mostly weak nonspecific interactions partly mediated through a water layer [51,52] Such protein–DNA interactions are easily competed out by small cations [53] Because the initial recognition complexes exist for longer during processing of 8-oxoG:A by either Fpg or OGG1, whereas with 8-oxoG:C the reaction quickly proceeds to its catalytic steps [19,20], the effect of electrostatic screening by higher ionic strength may be more pronounced with 8-oxoG:A substrates; it is also possible that electrostatic interactions may stabilize catalytically inactive conformations of the enzyme in the case of 8-oxoG:A Unlike 8-oxoG:C pairs that exist in a conventional anti ⁄ anti conformation [54], the 8-oxoG deoxynucleotide in 8-oxoG:A mispairs prefers a syn conformation due to steric repulsion between the O8 atom and the 5¢-phosphate [3,55] Thermodynamic and modeling studies suggest that 8-oxoG may exist in a syn ⁄ anti equilibrium when paired with A [56,57] Mg2+ is known to induce conformational transitions in nucleic acids, possibly by selective stabilization ⁄ destabilization of one of the conformations; for example, submillimolar concentrations of Mg2+ induce a transition of poly(dGm5dC)Ỉpoly(dG-m5dC) from the B-DNA to the Z-DNA form [58] Thus, the apparent increase in the C ⁄ A specificity of Fpg and OGG1 in the presence of MgCl2 may be caused by a shift in the conformational equilibrium of the 8-oxoG:A pair towards 8-oxoG(syn):A(anti), which may be poorly recognized by the enzyme [23] In the case of OGG1, another possible mode of Mg2+ action may be via binding to a metal-binding site formed at the OGG1 ⁄ DNA interface [16], with a potential to destabilize the catalytically competent conformation of the enzyme–substrate complex with an adenine base opposite the lesion The effect of magnesium may be aggravated by the tendency of its solvated ions to form multiple water bridges with adjacent positions in DNA [59], possibly influencing conformational dynamics of damaged DNA during recognition and catalysis by Fpg 3756 or OGG1 Regarding OGG1, our results are in agreement with a recent report [60] that a high Mg2+ concentration inhibits the AP lyase activity of the enzyme more than its DNA glycosylase activity The nature of anions in the reaction mixture did not affect the general specificity, although some details were notably different between reactions performed in the presence of KCl and KGlu For example, KGlu supported the DNA glycosylase activity of OGG1 on 8-oxoG:A over a much wider range of salt and Mg2+ concentrations than KCl did, and tended to sustain the activity of Fpg and the AP lyase activity of OGG1 at low Mg2+ better than KCl At the same time, high KGlu concentrations selectively decreased the turnover of OGG1 on the 8-oxoG:A substrate and the affinity of OGG1 for the uncleavable THF:A ligand Some anions are known to influence the activity of DNA glycosylases; for example, phosphate is a competitive inhibitor of Fpg with Ki $ 10 mm [61], and we observed that the activity of Fpg on 8-oxoG:C in the absence of KPi was generally higher than in its presence However, it seems that the overall differences between Cl) and glutamate are not decisive for the opposite-base specificity of Fpg and OGG1 Also, although the buffering capacity of KGlu at pH 7.5 is much weaker than that of KPi, the omission of KPi had a rather minor effect on the specificity of both enzymes Even less important for the activity and opposite-base specificity of Fpg and OGG1 were polyamines (spermine and spermidine), crowding agents [poly(ethylene glycol)], and small heterocyclic molecules (biotin and caffeine) The effect that polyamines and crowding agents may have on the activity of DNA-dependent enzymes in the cell is often under-appreciated, and some enzymes may be notably activated or inhibited by these factors [35,36,39,40] However, this is apparently not the case with Fpg and OGG1, and the kinetic parameters determined in the absence of polyamines and crowding agents need not be corrected when considering the intracellular environment Biotin and caffeine were selected for study as molecules that may, in principle, resemble purine substrates of Fpg and OGG1 and be bound in the active sites of these enzymes Modulation of the activity of DNA glycosylases by small, naturally encountered heterocyclic molecules is not without precedent: several DNA glycosylases are inhibited or activated by their substrate bases in a free form [62–65], other nucleobases [66] and even by heterocyclic molecules only distantly resembling the target bases (e.g MPG is inhibited by pterine) [65] Again, this was not observed with Fpg, which is also not inhibited by free 8-oxoG [21,60], and OGG1, which has been reported to be inhibited by 8-oxoG [60] FEBS Journal 275 (2008) 3747–3760 ª 2008 The Authors Journal compilation ª 2008 FEBS V S Sidorenko et al The problem of discriminating against the DNA elements that are close to the substrates but should not be processed is not unique for DNA glycosylases Wellknown phenomena are the misinsertion of an incorrect dNMP by DNA polymerases [67,68] or ‘star activity’ of restriction endonucleases [69] In all these cases, the fidelity of the enzymatic reaction may be significantly influenced by the presence of various compounds in the reaction mixture Generally, lower ionic strength, high crowding and divalent cations other than Mg2+ tend to facilitate non-specific reactions, whereas high ionic strength and Mg2+ increase the fidelity In this respect, some intracellular factors (ionic composition) favor correct, physiologically relevant substrates, whereas the others (crowding) act in the opposite direction As our results indicate, inside the cell, the preference of Fpg and OGG1 for processing substrates with C as compared with those containing A opposite the lesion, significantly relies on the high intracellular ionic strength and Mg2+ concentrations Experimental procedures Enzymes and oligonucleotides Fpg and OGG1 were overexpressed in E coli and purified as described [17,70] The concentrations of the active enzymes were determined by borohydride trapping and burst-phase kinetics as described previously [19,31] Phage T4 polynucleotide kinase was purchased from New England Biolabs (Beverly, MA, USA) Modified oligonucleotides d(CCTTCXCTCCTT) (X = THF) and d(CTCTCCCTTC XCTCCTTTCCTCT) (X = 8-oxoG) and complementary oligonucleotides placing C or A opposite the lesion were synthesized from commercially available phosphoramidites (Glen Research, Sterling, VA, USA) and purified by reverse-phase HPLC on a PRP-1 column (Hamilton, Reno, NV, USA) The modified strand was 32P-labeled using T4 polynucleotide kinase, purified by reverse-phase chromatography on a NenSorb C18 sorbent (Applera, Norwalk, CT, USA) according to the manufacturer’s instructions, and annealed to a twofold molar excess of the appropriate complementary strand Cleavage assay The standard reaction mixture [10 lL, 20 lL if poly(ethylene glycol) was present] included 50 nm 32P-labeled duplex substrate, 25 mm KPi (pH 7.5; omitted in some specified cases), mm dithiothreitol and Fpg (5 nm) or OGG1 (10 nm for the glycosylase activity assay, 100 nm for the AP lyase activity assay) The enzymes were diluted to 10-fold working concentrations in 12.5 mm KPi (pH 7.5) supplemented with 0.5 mgỈmL)1 BSA The varied compo- Factors affecting the specificity of Fpg and OGG1 nents of the reaction mixture included KCl, KGlu, MgCl2, spermine or spermidine and poly(ethylene glycol) (4000 or 8000); the components included in complete factorial design experiments are summarized in Table When KGlu was used as a component of the reaction mixture, its stock was first brought to pH 7.5 by titration with glutamic acid and then used to prepare the reaction mixtures; the concentrations are reported with respect to glutamate anion If necessary, the reaction mixture was supplemented with 0.1–20 mm biotin or caffeine The reaction was initiated by adding the enzyme (1 ⁄ 10 volume of the reaction mixture) and allowed to proceed for (Fpg) or 10 (OGG1) In the case of Fpg, the reaction was terminated by mixing with ⁄ vol of formamide loading dye [71] and heating for at 95 °C To measure the glycosylase activity of OGG1, the reaction was terminated by heating for 30 at 95 °C (AP sites are fully cleaved under these conditions) [72], mixed with ⁄ vol of formamide loading dye and heated again for at 95 °C To measure the AP lyase activity of OGG1, the reaction was terminated by mixing with ⁄ vol of formamide loading dye followed by heating for at 95 °C and storage on ice until analysis The reaction products were separated by denaturing PAGE (20% polyacrylamide, 7.2 m urea) and quantified using a Molecular Imager FX system (Bio-Rad, Hercules, CA, USA) Kinetic constant determination The reaction mixtures were as in the standard assay, except that the components specified in the Results were added, and the concentrations of the substrate and enzyme varied as required for the respective kinetics To calculate the steadystate catalytic constants (KM and kcat) for Fpg, the enzyme was used at nm (8-oxoG:C) or 10 nm (8-oxoG:A) and the substrate concentrations varied from 1.5 to 100 nm (8-oxoG:C) or from 40 to 2700 nm (8-oxoG:A) To determine the base excision rate constant (k2) for OGG1 in the single turnover assay, the reaction mixture included 10 nm substrate and 100 or 2000 nm enzyme (for 8-oxoG:C and 8-oxoG:A, respectively) The reaction mixture was incubated at °C for 0.5–30 (8-oxoG:C) or 1–90 (8-oxoG:A) and the glycosylase activity was assayed as above To determine the product release rate constant (k3) for OGG1 in the burst rate assay, the reaction mixture included 50 nm substrate and 10 nm enzyme The reaction mixture was incubated at 37 °C for 0.5–5 and the glycosylase activity was assayed All reported kinetic constants were obtained by non-linear least-square fitting using sigmaplot v 8.0 software (Systat Software, San Jose, CA, USA) Fluorimetric titration Binding of Fpg and OGG1 to the THF-containing ligands was assayed by following tryptophan fluorescence of the enzymes using an SFM-25 spectrofluorimeter (Kontron, FEBS Journal 275 (2008) 3747–3760 ª 2008 The Authors Journal compilation ª 2008 FEBS 3757 Factors affecting the specificity of Fpg and OGG1 V S Sidorenko et al Munich, Germany), equipped with a 80 lL cuvette thermostated at 15 °C The fluorescence was excited at kex = 290 nm, and the emission was recorded at kem = 300– 360 nm The reaction mixtures were the same as described under ‘Kinetic constants determination’ except the enzyme concentrations were different (1 lm Fpg, 3.6 lm OGG1) and the oligonucleotide ligand was added in small increments until the binding curve was saturated Kd values were extracted by fitting the fluorescence data to a single-site ligand-binding model [70] 10 11 Acknowledgments This work was supported in part by grants from the Presidium of the Russian Academy of Sciences (Program 10.1), Russian Foundation for Basic Research (07-04-00395, 08-04-00596), Russian Ministry of Science and Education (02.512.11.2194), and an Integrative Grant from the Siberian Branch of the Russian Academy of Sciences (Program 6) We thank Dr N A Kuznetsov (Institute of Chemical Biology and Fundamental Medicine) for help with collecting the fluorescence data 12 13 References Lindahl T (1993) Instability and decay of the primary structure of DNA Nature 362, 709–715 European Standards Committee on Oxidative DNA Damage (ESCODD), Gedik CM & Collins A (2005) Establishing the background level of base oxidation in human lymphocyte DNA: results of an interlaboratory validation study FASEB J 19, 82–84 Kouchakdjian M, Bodepudi V, Shibutani S, Eisenberg M, Johnson F, Grollman AP & Patel DJ (1991) NMR structural studies of the ionizing radiation adduct 7-hydro-8-oxodeoxyguanosine (8-oxo-7H-dG) opposite deoxyadenosine in a DNA duplex 8-Oxo-7H-dG(syn)Ỉ dA(anti) alignment at lesion site Biochemistry 30, 1403– 1412 Shibutani S, Takeshita M & Grollman AP (1991) Insertion of specific bases during DNA synthesis past the oxidation-damaged base 8-oxodG Nature 349, 431– 434 Hollstein M, Sidransky D, Vogelstein B & Harris CC (1991) p53 mutations in human cancers Science 253, 49–53 Li D, Firozi PF, Zhang W, Shen J, DiGiovanni J, Lau S, Evans D, Friess H, Hassan M & Abbruzzese JL (2001) DNA adducts, genetic polymorphisms, and K-ras mutation in human pancreatic cancer Mutat Res 513, 37–48 Michaels ML & Miller JH (1992) The GO system protects organisms from the mutagenic effect of the 3758 14 15 16 17 18 19 20 spontaneous lesion 8-hydroxyguanine (7,8-dihydro8-oxoguanine) J Bacteriol 174, 6321–6325 Tchou J & Grollman AP (1993) Repair of DNA containing the oxidatively damaged base, 8-oxoguanine Mutat Res 299, 277–287 Tsuzuki T, Nakatsu Y & Nakabeppu Y (2007) Significance of error-avoiding mechanisms for oxidative DNA damage in carcinogenesis Cancer Sci 98, 465–470 Wilson DM III & Bohr VA (2007) The mechanics of base excision repair, and its relationship to aging and disease DNA Repair 6, 544–559 Tajiri T, Maki H & Sekiguchi M (1995) Functional cooperation of MutT, MutM and MutY proteins in preventing mutations caused by spontaneous oxidation of guanine nucleotide in Escherichia coli Mutat Res 336, 257–267 Sakumi K, Tominaga Y, Furuichi M, Xu P, Tsuzuki T, Sekiguchi M & Nakabeppu Y (2003) Ogg1 knockoutassociated lung tumorigenesis and its suppression by Mth1 gene disruption Cancer Res 63, 902–905 Xie Y, Yang H, Cunanan C, Okamoto K, Shibata D, Pan J, Barnes DE, Lindahl T, McIlhatton M, Fishel R et al (2004) Deficiencies in mouse myh and ogg1 result in tumor predisposition and G to T mutations in codon 12 of the k-ras oncogene in lung tumors Cancer Res 64, 3096–3102 Al-Tassan N, Chmiel NH, Maynard J, Fleming N, Livingston AL, Williams GT, Hodges AK, Davies DR, David SS, Sampson JR et al (2002) Inherited variants of MYH associated with somatic G:C fi T:A mutations in colorectal tumors Nat Genet 30, 227–232 Weiss JM, Goode EL, Ladiges WC & Ulrich CM (2005) Polymorphic variation in hOGG1 and risk of cancer: a review of the functional and epidemiologic literature Mol Carcinogen 42, 127–141 Bruner SD, Norman DPG & Verdine GL (2000) Structural basis for recognition and repair of the endogenous mutagen 8-oxoguanine in DNA Nature 403, 859–866 Gilboa R, Zharkov DO, Golan G, Fernandes AS, Gerchman SE, Matz E, Kycia JH, Grollman AP & Shoham G (2002) Structure of formamidopyrimidineDNA glycosylase covalently complexed to DNA J Biol Chem 277, 19811–19816 Fromme JC & Verdine GL (2002) Structural insights into lesion recognition and repair by the bacterial 8-oxoguanine DNA glycosylase MutM Nat Struct Biol 9, 544–552 Kuznetsov NA, Koval VV, Zharkov DO, Vorobjev YN, Nevinsky GA, Douglas KT & Fedorova OS (2007) Pre-steady-state kinetic study of substrate specificity of Escherichia coli formamidopyrimidine-DNA glycosylase Biochemistry 46, 424–435 Kuznetsov NA, Koval VV, Nevinsky GA, Douglas KT, Zharkov DO & Fedorova OS (2007) Kinetic conforma- FEBS Journal 275 (2008) 3747–3760 ª 2008 The Authors Journal compilation ª 2008 FEBS V S Sidorenko et al 21 22 23 24 25 26 27 28 29 30 31 tional analysis of human 8-oxoguanine-DNA glycosylase J Biol Chem 282, 1029–1038 Tchou J, Kasai H, Shibutani S, Chung M-H, Laval J, Grollman AP & Nishimura S (1991) 8-Oxoguanine (8-hydroxyguanine) DNA glycosylase and its substrate specificity Proc Natl Acad Sci USA 88, 4690–4694 Castaing B, Geiger A, Seliger H, Nehls P, Laval J, Zelwer C & Boiteux S (1993) Cleavage and binding of a DNA fragment containing a single 8-oxoguanine by wild type and mutant FPG proteins Nucleic Acids Res 21, 2899–2905 Tchou J, Bodepudi V, Shibutani S, Antoshechkin I, Miller J, Grollman AP & Johnson F (1994) Substrate specificity of Fpg protein: recognition and cleavage of oxidatively damaged DNA J Biol Chem 269, 15318– 15324 Auffret van der Kemp P, Thomas D, Barbey R, de Oliveira R & Boiteux S (1996) Cloning and expression in Escherichia coli of the OGG1 gene of Saccharomyces cerevisiae, which codes for a DNA glycosylase that excises 7,8-dihydro-8-oxoguanine and 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine Proc Natl Acad Sci USA 93, 5197–5202 ˚ Bjøras M, Luna L, Johnsen B, Hoff E, Haug T, Rognes T & Seeberg E (1997) Opposite base-dependent reactions of a human base excision repair enzyme on DNA containing 7,8-dihydro-8-oxoguanine and abasic sites EMBO J 16, 6314–6322 Zharkov DO, Rosenquist TA, Gerchman SE & Grollman AP (2000) Substrate specificity and reaction mechanism of murine 8-oxoguanine-DNA glycosylase J Biol Chem 275, 28607–28617 Sundararaj S, Guo A, Habibi-Nazhad B, Rouani M, Stothard P, Ellison M & Wishart DS (2004) The CyberCell Database (CCDB): a comprehensive, self-updating, relational database to coordinate and facilitate in silico modeling of Escherichia coli Nucleic Acids Res 32, D293–D295 Bailly V, Verly WG, O’Connor T & Laval J (1989) Mechanism of DNA strand nicking at apurinic ⁄ apyrimidinic sites by Escherichia coli [formamidopyrimidine]DNA glycosylase Biochem J 262, 581–589 Lipanov A, Kopka ML, Kaczor-Grzeskowiak M, Quintana J & Dickerson RE (1993) Structure of the B-DNA decamer C-C-A-A-C-I-T-T-G-G in two different space groups: conformational flexibility of B-DNA Biochemistry 32, 1373–1389 Hill JW, Hazra TK, Izumi T & Mitra S (2001) Stimulation of human 8-oxoguanine-DNA glycosylase by AP-endonuclease: potential coordination of the initial steps in base excision repair Nucleic Acids Res 29, 430– 438 Sidorenko VS, Nevinsky GA & Zharkov DO (2007) Mechanism of interaction between human 8-oxo- Factors affecting the specificity of Fpg and OGG1 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 guanine-DNA glycosylase and AP endonuclease DNA Repair 6, 317–328 O’Connor TR, Graves RJ, de Murcia G, Castaing B & Laval J (1993) Fpg protein of Escherichia coli is a zinc finger protein whose cysteine residues have a structural and ⁄ or functional role J Biol Chem 268, 9063–9070 Paz-Elizur T, Takeshita M & Livneh Z (1997) Mechanism of bypass synthesis through an abasic site analog by DNA polymerase I Biochemistry 36, 1766–1773 Moinard C, Cynober L & de Bandt J-P (2005) Polyamines: metabolism and implications in human diseases Clin Nutr 24, 184–197 Kun E, Kirsten E, Mendeleyev J & Ordahl CP (2004) Regulation of the enzymatic catalysis of poly(ADPribose) polymerase by dsDNA, polyamines, Mg2+, Ca2+, histones H1 and H3, and ATP Biochemistry 43, 210–216 Bakhanashvili M, Novitsky E, Levy I & Rahav G (2005) The fidelity of DNA synthesis by human immunodeficiency virus type reverse transcriptase increases in the presence of polyamines FEBS Lett 579, 1435–1440 Male R, Fosse VM & Kleppe K (1982) Polyamineinduced hydrolysis of apurinic sites in DNA and nucleosomes Nucleic Acids Res 10, 6305–6318 Ellis RJ (2001) Macromolecular crowding: obvious but underappreciated Trends Biochem Sci 26, 597–604 Pheiffer BH & Zimmerman SB (1983) Polymer-stimulated ligation: enhanced blunt- or cohesive-end ligation of DNA or deoxyribooligonucleotides by T4 DNA ligase in polymer solutions Nucleic Acids Res 11, 7853–7871 Wenner JR & Bloomfield VA (1999) Crowding effects on EcoRV kinetics and binding Biophys J 77, 3234– 3241 Struthers L, Patel R, Clark J & Thomas S (1998) Direct detection of 8-oxodeoxyguanosine and 8-oxoguanine by avidin and its analogues Anal Biochem 255, 20–31 Conners R, Hooley E, Clarke AR, Thomas S & Brady RL (2006) Recognition of oxidatively modified bases within the biotin-binding site of avidin J Mol Biol 357, 263–274 Porta M, Vioque J, Ayude D, Alguacil J, Jariod M, Ruiz L & Murillo JA (2003) Coffee drinking: the rationale for treating it as a potential effect modifier of carcinogenic exposures Eur J Epidemiol 18, 289–298 Zharkov DO, Shoham G & Grollman AP (2003) Structural characterization of the Fpg family of DNA glycosylases DNA Repair 2, 839–862 Boiteux S, O’Connor TR & Laval J (1987) Formamidopyrimidine-DNA glycosylase of Escherichia coli: cloning and sequencing of the fpg structural gene and overproduction of the protein EMBO J 6, 3177–3183 Karakaya A, Jaruga P, Bohr VA, Grollman AP & Dizdaroglu M (1997) Kinetics of excision of purine lesions from DNA by Escherichia coli Fpg protein Nucleic Acids Res 25, 474–479 FEBS Journal 275 (2008) 3747–3760 ª 2008 The Authors Journal compilation ª 2008 FEBS 3759 Factors affecting the specificity of Fpg and OGG1 V S Sidorenko et al 47 Zharkov DO & Grollman AP (2005) The DNA trackwalkers: principles of lesion search and recognition by DNA glycosylases Mutat Res 577, 24–54 48 Adhikari S, Toretsky JA, Yuan L & Roy R (2006) Magnesium, essential for base excision repair enzymes, inhibits substrate binding of N-methylpurine-DNA glycosylase J Biol Chem 281, 29525–29532 49 Eide L, Luna L, Gustad EC, Henderson PT, Essigmann JM, Demple B & Seeberg E (2001) Human endonuclease III acts preferentially on DNA damage opposite guanine residues in DNA Biochemistry 40, 6653–6659 50 Radicella JP, Dherin C, Desmaze C, Fox MS & Boiteux S (1997) Cloning and characterization of hOGG1, a human homolog of the OGG1 gene of Saccharomyces cerevisiae Proc Natl Acad Sci USA 94, 8010–8015 51 Banerjee A, Yang W, Karplus M & Verdine GL (2005) Structure of a repair enzyme interrogating undamaged DNA elucidates recognition of damaged DNA Nature 434, 612–618 52 Banerjee A, Santos WL & Verdine GL (2006) Structure of a DNA glycosylase searching for lesions Science 311, 1153–1157 53 Berg OG, Winter RB & von Hippel PH (1981) Diffusion-driven mechanisms of protein translocation on nucleic acids Models and theory Biochemistry 20, 6929–6948 54 Lipscomb LA, Peek ME, Morningstar ML, Verghis SM, Miller EM, Rich A, Essigmann JM & Williams LD (1995) X-Ray structure of a DNA decamer containing 7,8-dihydro-8-oxoguanine Proc Natl Acad Sci USA 92, 719–723 55 McAuley-Hecht KE, Leonard GA, Gibson NJ, Thomson JB, Watson WP, Hunter WN & Brown T (1994) Crystal structure of a DNA duplex containing 8-hydroxydeoxyguanine-adenine base pairs Biochemistry 33, 10266–10270 56 Plum GE, Grollman AP, Johnson F & Breslauer KJ (1995) Influence of the oxidatively damaged adduct 8-oxodeoxyguanosine on the conformation, energetics, and thermodynamic stability of a DNA duplex Biochemistry 34, 16148–16160 57 Cheng XL, Kelso C, Hornak V, de los Santos C, Grollman AP & Simmerling C (2005) Dynamic behavior of DNA base pairs containing 8-oxoguanine J Am Chem Soc 127, 13906–13918 58 Behe M & Felsenfeld G (1981) Effects of methylation on a synthetic polynucleotide: the B–Z transition in 3760 59 60 61 62 63 64 65 66 67 68 69 70 71 72 poly(dG-m5dC)Ỉpoly(dG-m5dC) Proc Natl Acad Sci USA 78, 1619–1623 ´ Subirana JA & Soler-Lopez M (2003) Cations as hydrogen bond donors: a view of electrostatic interactions in DNA Annu Rev Biophys Biomol Struct 32, 27–45 ˚ Morland I, Luna L, Gustad E, Seeberg E & Bjøras M (2005) Product inhibition and magnesium modulate the dual reaction mode of hOgg1 DNA Repair 4, 381–387 Ishchenko AA, Vasilenko NL, Sinitsina OI, Yamkovoy VI, Fedorova OS, Douglas KT & Nevinsky GA (2002) Thermodynamic, kinetic, and structural basis for recognition and repair of 8-oxoguanine in DNA by Fpg protein from Escherichia coli Biochemistry 41, 7540–7548 Krokan H & Wittwer CU (1981) Uracil DNA-glycosylase from HeLa cells: general properties, substrate specificity and effect of uracil analogs Nucleic Acids Res 9, 2599–2613 Thomas L, Yang CH & Goldthwait DA (1982) Two DNA glycosylases in Escherichia coli which release primarily 3-methyladenine Biochemistry 21, 1162–1169 Bjelland S & Seeberg E (1987) Purification and characterization of 3-methyladenine DNA glycosylase I from Escherichia coli Nucleic Acids Res 15, 2787–2801 Roy R, Brooks C & Mitra S (1994) Purification and biochemical characterization of recombinant N-methylpurine-DNA glycosylase of the mouse Biochemistry 33, 15131–15140 Fromme JC, Bruner SD, Yang W, Karplus M & Verdine GL (2003) Product-assisted catalysis in base-excision DNA repair Nat Struct Biol 10, 204–211 Kunkel TA & Bebenek K (2000) DNA replication fidelity Annu Rev Biochem 69, 497–529 Kool ET (2002) Active site tightness and substrate fit in DNA replication Annu Rev Biochem 71, 191–219 Pingoud A & Jeltsch A (1997) Recognition and cleavage of DNA by type-II restriction endonucleases Eur J Biochem 246, 1–22 Sambrook J & Russell DW (2001) Molecular Cloning: A Laboratory Manual, 3rd edn Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY Zharkov DO & Grollman AP (1998) MutY DNA glycosylase: base release and intermediate complex formation Biochemistry 37, 12384–12394 Kuznetsov NA, Koval VV, Zharkov DO, Nevinsky GA, Douglas KT & Fedorova OS (2005) Kinetics of substrate recognition and cleavage by human 8-oxoguanine-DNA glycosylase Nucleic Acids Res 33, 3919– 3931 FEBS Journal 275 (2008) 3747–3760 ª 2008 The Authors Journal compilation ª 2008 FEBS ... evaluate the effects of ionic strength and Mg2+ on the activity and specificity of OGG1, we measured the apparent values of k2 and k3 under conditions of low salt (KPi only) and no Mg2+, low salt and. .. Effects of ionic strength and divalent cations on the activity and specificity of Fpg and OGG1 The conditions inside a living cell differ from most buffer systems in which the activity and specificity. .. may be affected by ionic strength, the concentration of divalent cations such as Mg2+, the nature of the buffering agents, the presence of competing polyamines and other small molecules, and crowding