Portal de Periódicos da CAPES

The Escherichia coli 3-Methyladenine DNA Glycosylase AlkA Has a Remarkably Versatile Active Site

2004; Elsevier BV; Volume: 279; Issue: 26 Linguagem: Inglês

10.1074/jbc.m403860200

1083-351X

Patrick O’Brien, Tom Ellenberger,

CRISPR and Genetic Engineering

3-Methyladenine DNA glycosylase II (AlkA) from Escherichia coli is induced in response to DNA alkylation, and it protects cells from alkylated nucleobases by catalyzing their excision. In contrast to the highly specific 3-methyladenine DNA glycosylase I (E. coli TAG) that catalyzes the excision of 3-methyl adducts of adenosine and guanosine from DNA, AlkA catalyzes the excision of a wide variety of alkylated bases including N-3 and N-7 adducts of adenosine and guanosine and O2 adducts of thymidine and cytidine. We have investigated how AlkA can recognize a diverse set of damaged bases by characterizing its discrimination between oligonucleotide substrates in vitro. Similar rate enhancements are observed for the excision of a structurally diverse set of substituted purine bases and of the normal purines adenine and guanine. These results are consistent with a remarkably indiscriminate active site and suggest that the rate of AlkA-catalyzed excision is dictated not by the catalytic recognition of a specific substrate but instead by the reactivity of the N-glycosidic bond of each substrate. Damaged bases with altered base pairing have a modest advantage, as mismatches are processed up to 400-fold faster than stable Watson-Crick base pairs. Nevertheless, AlkA does not effectively exclude undamaged DNA from its active site. The resulting deleterious excision of normal bases is expected to have a substantial cost associated with the expression of AlkA. 3-Methyladenine DNA glycosylase II (AlkA) from Escherichia coli is induced in response to DNA alkylation, and it protects cells from alkylated nucleobases by catalyzing their excision. In contrast to the highly specific 3-methyladenine DNA glycosylase I (E. coli TAG) that catalyzes the excision of 3-methyl adducts of adenosine and guanosine from DNA, AlkA catalyzes the excision of a wide variety of alkylated bases including N-3 and N-7 adducts of adenosine and guanosine and O2 adducts of thymidine and cytidine. We have investigated how AlkA can recognize a diverse set of damaged bases by characterizing its discrimination between oligonucleotide substrates in vitro. Similar rate enhancements are observed for the excision of a structurally diverse set of substituted purine bases and of the normal purines adenine and guanine. These results are consistent with a remarkably indiscriminate active site and suggest that the rate of AlkA-catalyzed excision is dictated not by the catalytic recognition of a specific substrate but instead by the reactivity of the N-glycosidic bond of each substrate. Damaged bases with altered base pairing have a modest advantage, as mismatches are processed up to 400-fold faster than stable Watson-Crick base pairs. Nevertheless, AlkA does not effectively exclude undamaged DNA from its active site. The resulting deleterious excision of normal bases is expected to have a substantial cost associated with the expression of AlkA. The reactivity of nucleobases in DNA renders their spontaneous alkylation by cellular metabolites unavoidable, and exposure to exogenous alkylating agents greatly increases the amount of DNA damage (for review, see Refs. 1Lindahl T. Nature. 1993; 362: 709-715Crossref PubMed Scopus (4378) Google Scholar and 2Sedgwick B. Nat. Rev. Mol. Cell. Biol. 2004; 5: 148-157Crossref PubMed Scopus (287) Google Scholar). Alkylated bases block DNA-templated activities such as replication and transcription, and they cause mutations during DNA replication. The efficient repair of alkyl base adducts is complicated by their chemical diversity. For example, purines can be alkylated at positions N-1, N-3, and N-7 of the purine ring and at the exocyclic O6 of guanine, and pyrimidines can be alkylated on O2 of cytosine and thymine or O4 of thymine. An elaborate DNA repair response has evolved to process these diverse lesions, either by the direct reversal of alkylation or more commonly via base excision repair (2Sedgwick B. Nat. Rev. Mol. Cell. Biol. 2004; 5: 148-157Crossref PubMed Scopus (287) Google Scholar). The base excision repair pathway is initiated by DNA repair glycosylases that locate damaged bases within genomic DNA and catalyze the hydrolysis of the N-glycosidic bond to release the damaged base, resulting in the formation of an abasic site. Completion of the repair pathway requires the subsequent action of an abasic site-specific endonuclease, a deoxyribophosphodiesterase, a DNA polymerase, and a DNA ligase. The DNA glycosylases that initiate repair expose substrate nucleotides in double-stranded DNA by the process of base flipping (3Hollis T. Ichikawa Y. Ellenberger T. EMBO J. 2000; 19: 758-766Crossref PubMed Scopus (206) Google Scholar, 4Roberts R.J. Cheng X. Annu. Rev. Biochem. 1998; 67: 181-198Crossref PubMed Scopus (308) Google Scholar). Enzymatic specificity could be manifested by preferential binding to damaged DNA, by differential base flipping of damaged nucleotides, or by selective engagement of the active site with flipped-out substrates that are damaged. In Escherichia coli two alkylation-specific DNA glycosylases have been identified that catalyze the excision of cytotoxic 3-methyladenine lesions (5Thomas L. Yang C.-H. Goldthwait D.A. Biochemistry. 1982; 21: 1162-1169Crossref PubMed Scopus (111) Google Scholar). 3-Methyladenine DNA glycosylase I (TAG), 1The abbreviations used are: TAG, 3-methyladenine DNA glycosylase I; AAG, human alkyladenine DNA glycosylase (also known as MPG); AlkA, 3-methyladenine DNA glycosylase II; P, purine; 3mA, 3-methyladenine; 7mG, 7-methylguanine; ϵA, 1,N6-ethenoadenine; Hx, hypoxanthine; Pyr, pyrrolidine; Aza, 1-azaribose. the product of the tag gene, is constitutively expressed and has a narrow substrate range. TAG catalyzes the excision of 3-alkyl-substituted adenosine or guanosine, but it does not recognize other alkylated bases (6Bjelland S. Bjøras M. Seeberg E. Nucleic Acids Res. 1993; 21: 2045-2049Crossref PubMed Scopus (79) Google Scholar). 3-Methyladenine DNA glycosylase II, encoded by the alkA gene, is normally expressed at low levels and up-regulated following exposure to DNA alkylating agents as part of the adaptive response (7Samson L. Caims J. Nature. 1977; 267: 281-283Crossref PubMed Scopus (569) Google Scholar, 8Evensen G. Seeberg E. Nature. 1982; 296: 773-775Crossref PubMed Scopus (222) Google Scholar, 9Nakabeppu Y. Miyata T. Kondo H. Iwanaga S. Sekiguchi M. J. Biol. Chem. 1984; 259: 13730-13736Abstract Full Text PDF PubMed Google Scholar). AlkA has a very broad substrate range, catalyzing the excision of N-3- and N-7-alkyl purines as well as O2-alkyl pyrimidines (6Bjelland S. Bjøras M. Seeberg E. Nucleic Acids Res. 1993; 21: 2045-2049Crossref PubMed Scopus (79) Google Scholar, 10Bjelland S. Birkeland N. Benneche T. Volden G. Seeberg E. J. Biol. Chem. 1994; 269: 30489-30495Abstract Full Text PDF PubMed Google Scholar). In addition to these common alkyl adducts, AlkA has been shown to excise such disparate lesions as the cyclic adducts ϵA and ϵC (3,N4-ethenocytosine), deaminated bases such as hypoxanthine and xanthosine, and the oxidative lesions oxanine and 5-formyluracil (11Saparbaev M. Kleibl K. Laval J. Nucleic Acids Res. 1995; 23: 3750-3755Crossref PubMed Scopus (214) Google Scholar, 12Saparbaev M. Laval J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5873-5877Crossref PubMed Scopus (234) Google Scholar, 13Masaoka A. Terato H. Kobayashi M. Honsho A. Ohyama Y. Ide H. J. Biol. Chem. 1999; 274: 25136-25143Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 14Terato H. Masaoka A. Asagoshi K. Honsho A. Ohyama Y. Suzuki T. Yamada M. Makino K. Yamamoto K. Ide H. Nucleic Acids Res. 2002; 30: 4975-4984Crossref PubMed Scopus (51) Google Scholar). AlkA homologs are found in many prokaryotic and eukaryotic organisms, but in plants and vertebrates this enzyme is replaced with another broadly specific DNA glycosylase AAG. This broad substrate range of AlkA is remarkable, and it raises the question of whether such a diverse range of DNA lesions can actually be recognized as being different from the vast excess of normal, unmodified bases. The broad substrate range of AlkA differs markedly from other well characterized DNA glycosylases such as those that are specific for uracil, 8-oxoguanine, thymine, and adenine. In each case, specific binding interactions allow the damaged base or bases to be distinguished from the undamaged bases in DNA. The broad specificity of AlkA is at odds with the discrimination against undamaged bases, which are generally smaller than alkylated substrates. AlkA has been shown to have low levels of activity for the excision of each of the normal bases from DNA (15Berdal K.G. Johansen R.F. Seeberg E. EMBO J. 1998; 17: 363-367Crossref PubMed Scopus (156) Google Scholar). The deleterious excision of undamaged bases is a likely explanation for the toxicity or increased mutation rate that is associated with the overexpression of AlkA or its yeast homolog Mag1 (15Berdal K.G. Johansen R.F. Seeberg E. EMBO J. 1998; 17: 363-367Crossref PubMed Scopus (156) Google Scholar, 16Glassner B.J. Rasmussen L.J. Najarian M.T. Posnick L.M. Samson L.D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9997-10002Crossref PubMed Scopus (176) Google Scholar, 17Posnick L.M. Samson L.D. J. Bacteriol. 1999; 181: 6763-6771Crossref PubMed Google Scholar). We have characterized the glycosylase activity of AlkA toward a variety of damaged and undamaged bases in defined oligonucleotides. We find that AlkA prefers to excise bases from nucleotides that are mispaired. Damaged bases that interfere with base pairing in DNA are more readily flipped-out into the active site, providing some selectivity for excision of the damaged base. A comparison of the rate enhancements for excision of structurally disparate bases reveals a remarkably nonspecific active site that can accommodate a broad range of substrate bases (15Berdal K.G. Johansen R.F. Seeberg E. EMBO J. 1998; 17: 363-367Crossref PubMed Scopus (156) Google Scholar). Indeed, we find that the preferential excision of alkylated bases can be quantitatively explained by the decreased N-glycosidic bond stability of N-alkylated bases. The poor discrimination between damaged and undamaged bases by AlkA is manifested by the frequent excision of undamaged bases, providing an explanation for why expression of AlkA is tightly repressed under normal growth conditions. However, such a broadly specific enzyme may offer an evolutionary advantage because it is immediately available to process new types of DNA damage before a specific response can evolve. DNA substrates of the sequence 5′-CGATAGCATCCTXCCTTCTCTCCAT annealed to the complementary oligonucleotide 5′-ATGGAGAGAAGGYAGGATGCTATCG, in which lesion X is paired with base Y (X·Y), were prepared as described previously (18O'Brien P.J. Ellenberger T. Biochemistry. 2003; 42: 12418-12429Crossref PubMed Scopus (95) Google Scholar). DNA inhibitors of the sequence 5′-GACTACTACATGZTTGCCTACCTT annealed to the complementary oligonucleotide 5′-AAGGTAGGCAACCATGTAGTAGTC were prepared, in which Z was 1-azaribose (Aza (3Hollis T. Ichikawa Y. Ellenberger T. EMBO J. 2000; 19: 758-766Crossref PubMed Scopus (206) Google Scholar, 19Deng L. Schärer O.D. Verdine G.L. J. Am. Chem. Soc. 1997; 119: 7865-7866Crossref Scopus (59) Google Scholar)) or pyrrolidine (Pyr (20Schärer O.D. Nash H.M. Jirieny J. Laval J. Verdine G.L. J. Biol. Chem. 1998; 273: 8592-8597Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 21Lau A.Y. Schärer O.D. Samson L. Verdine G.L. Ellenberger T. Cell. 1998; 95: 249-258Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar)). Wild-type and mutant (D238N) AlkA proteins were overexpressed in E. coli and purified as described previously (22Labahn J. Schärer O.D. Long A. Ezaz-Nikpay K. Verdine G.L. Ellenberger T.E. Cell. 1996; 88: 321-329Abstract Full Text Full Text PDF Scopus (232) Google Scholar). The protein concentration was determined by absorbance at 280 nm using the calculated extinction coefficient of 6.7 × 104m–1 cm–1. General Kinetic Methods—Glycosylase activity was measured using a 32P-based assay. Single-stranded oligonucleotides were 5′-labeled with T4 polynucleotide kinase, annealed to a complementary oligonucleotide, and incubated with AlkA. Reactions were quenched by the addition of sodium hydroxide (0.2 m final), and abasic DNA sites were subsequently cleaved by heating (0.2 m sodium hydroxide, 70 °C, 10 min). Samples were mixed with formamide loading buffer and resolved by denaturing PAGE. Product and substrate bands were quantified with a phosphorimaging system (Fuji BAS1000), and the fractional extent of reaction was monitored as a function of time. Enzymatic rate constants were obtained from exponential fits to the data (F = 1 – e–k×t) in which F is the fraction of product, t is time, and k is the observed rate constant. To ensure single-turnover conditions, the concentration of AlkA was kept in excess of the concentration of DNA. For the determination of kst the concentration of enzyme was varied over a range at least 10-fold above the K½ to ensure that the maximal rate constant was obtained. The rate constants for the slowest reactions (k ≤ 5 × 10–3 min–1) were obtained from a linear fit to the first 10% of the reaction (F = k × t). Unless otherwise stated, the standard reaction conditions were 37 °C with 50 mm sodium acetate, pH 6.0, 1 mm EDTA, 1 mm dithiothreitol, 0.1 mg/ml bovine serum albumin and ionic strength adjusted to 100 mm with sodium chloride. Although the pH value of 6.0 is below physiological pH, it is the observed pH optimum for many of the neutral purine substrates examined (see supplemental material). The reported rate enhancements are expected to be pH-independent because spontaneous and AlkA-catalyzed depurination have the same pH dependence between pH 6 and 8. Glycosylase Activity toward Purine-containing Mismatches—Given the similar rate constants for AlkA-catalyzed excision of the normal purines, and the greatly reduced activity of AlkA toward a nucleotide opposite an abasic site, it was necessary to consider the activity of AlkA toward both strands of a mismatch. This was achieved by individually labeling either of the two strands so that the rate constant and end point of the reaction could be independently obtained for AlkA-catalyzed excision from either strand. The results showed that excision of one base strongly inhibited the subsequent excision of the opposing base. As only a single base was excised from a given mismatch on the time scale of the assay, the observed rate constant is the sum of the individual rate constants for excision of either site. The rate constant for excision of a specific base is obtained by multiplying the observed rate constant by the end point of the reaction (kst = kobs × end point; see supplemental material). DNA Binding Assays—DNA binding affinities were determined by measuring the change in fluorescence anisotropy of DNA duplexes in which one strand was labeled with fluorescein at either the 5′ or 3′ end. The data were collected at 25 °C with a C-60 spectrofluorometer (Photon Technology International) with excitation and emission wave-lengths of 495 and 520 nm, respectively. The slit widths were typically 6 nm. Sample volumes varied between 150 μl (2-mm cuvette) and 1.5 ml (10-mm cuvette) depending upon the concentration of DNA required to obtain satisfactory signal to noise. To ensure that equilibrium binding constants were measured, the concentration of DNA was kept at least 10-fold below the observed Kd for binding, and the concentration of enzyme was varied over the range from 5-fold below to 5-fold above the Kd. Under these conditions the dissociation constants were calculated by fitting the model for a single binding site to the data (Fbound = [E]/(Kd + [E]), in which the [E] refers to the total concentration of protein and Fbound is the fraction of DNA bound). For the tight binding DNA inhibitors these conditions could not be satisfied, and the concentration of DNA was within 3–5-fold of the Kd for DNA binding. In these cases the concentration of DNA had to be considered (Fbound = {[DNA] + [E] + Kd – (([DNA] + [E] + Kd)2 – 4[DNA][E])½}/2[DNA]). In all cases DNA binding appeared to be rapid, as no difference in anisotropy was detected for incubation times between 2 and 20 min. We report macroscopic binding constants for oligonucleotide duplexes. However, the minimal site size for AlkA binding to DNA is expected to be ∼10 base pairs based upon the crystal structure of AlkA bound to DNA (3Hollis T. Ichikawa Y. Ellenberger T. EMBO J. 2000; 19: 758-766Crossref PubMed Scopus (206) Google Scholar), and thus there are ∼16 overlapping nonspecific binding sites on each 25-mer. The preferential excision of normal bases from near the end of the DNA raises the possibility that AlkA preferentially binds near DNA ends and that it might show slight preference for specific sequence contexts. The concentration of active protein was determined by direct titration with a high concentration of DNA (i.e. [DNA] » Kd). Under these conditions both wild-type and D238N AlkA were between 80 and 100% active, and only a single protein molecule bound to the 24-mer oligonucleotide duplex (see supplemental material). The similar anisotropy values observed for tight binding inhibitors and for weaker binding substrates are consistent with one molecule of AlkA binding per 25-mer substrate. AlkA-catalyzed Excision of 7-Methylguanine—We sought to quantify and dissect the substrate specificity of AlkA to understand the physical basis for how a broad substrate specificity can be achieved. To measure the specificity for damaged DNA we characterized the AlkA-catalyzed base excision of both damaged and undamaged bases. 7-Methylguanosine can be sitespecifically incorporated into a defined oligonucleotide, so we first characterized the AlkA-catalyzed reaction toward a 25-mer oligonucleotide duplex containing a single 7-methylguanosine lesion (23Asaeda A. Ide H. Asagoshi K. Matsuyama S. Tano K. Murakami A. Takamori Y. Kubo K. Biochemistry. 2000; 39: 1959-1965Crossref PubMed Scopus (50) Google Scholar). Like 3-methyladenosine, this lesion bears a positive charge (Fig. 1), and it has a greatly destabilized N-glycosidic bond. The activity of AlkA toward 7-methylguanosine and other lesions was compared with the activity toward undamaged oligonucleotides to provide a measure of the specificity for DNA damage. Many DNA glycosylases are inhibited by their product, an abasic site in DNA, so we measured single-turnover base excision kinetics. The single-turnover rate constant with saturating amounts of AlkA (kst) is analogous to the rate constant for multiple-turnover (kcat), but it does not include any of the steps associated with product release (Fig. 2). Single-turnover excision of 7-methylguanine (7mG) by AlkA follows a single exponential with a rate constant of 1.2 min–1 (data not shown). This rate constant is 1–2 orders of magnitude greater than the kcat values that have been previously reported for AlkA (0.01–0.07 min–1 (14Terato H. Masaoka A. Asagoshi K. Honsho A. Ohyama Y. Suzuki T. Yamada M. Makino K. Yamamoto K. Ide H. Nucleic Acids Res. 2002; 30: 4975-4984Crossref PubMed Scopus (51) Google Scholar, 24Gasparutto D. Dherin C. Boiteux S. Cadet J. DNA Rep. 2002; 1: 437-447Crossref PubMed Scopus (21) Google Scholar)), presumably because of the slow rate of product release that affects the steady-state rates. The single-turnover excision of 7mG catalyzed by AlkA showed a K½ value of ∼200 nm, which is larger than the previously reported Km values for multiple-turnover excision. Presumably the Km for multiple turnover includes a contribution from binding with high affinity to the abasic product. This rate constant for single-turnover excision of 7mG is similar to the rate constant for steady-state excision of 3mA (kcat = 0.5 min–1 (12Saparbaev M. Laval J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5873-5877Crossref PubMed Scopus (234) Google Scholar)). Opposing Base Specificity of AlkA and the Involvement of the Base Flipping Step in Substrate Selection—The relative ease of base flipping can significantly affect the activity of DNA glycosylases (25Biswas T. Clos L.J. SantaLucia J. Mitra S. Roy R. J. Mol. Biol. 2002; 320: 503-513Crossref PubMed Scopus (25) Google Scholar, 26O'Brien P.J. Ellenberger T. J. Biol. Chem. 2004; 279: 9750-9757Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 27Wyatt M.D. Samson L.D. Carcinogenesis. 2000; 21: 901-908Crossref PubMed Scopus (28) Google Scholar). To test whether the favorable base pairing of 7mG·C hinders AlkA-catalyzed glycosylase activity we compared the ability of AlkA to excise 7mG from different mismatched base pairs. AlkA does exhibit greater activity toward mismatched base pairs, with complete excision of 7mG occurring within a few seconds for 7mG·T and 7mG·A mismatches (data not shown). To more accurately measure these rates, the experiments were repeated at a lower temperature (Table I). AlkA is ∼25-fold more efficient at removing 7mG from either 7mG·T or 7mG·A mismatches than from a 7mG·C Watson-Crick base pair. The natural 7mG lesion occurs in 7mG·C base pairs and is therefore not a particularly good substrate for AlkA. These opposing base effects are consistent with the expected base pairing stability of 7mG and suggest that stable base pairing provides a barrier to substrate exposure.Table IEffect of the opposing base on the AlkA-catalyzed glycosylase reactionRelative rate constants for base excisionaFor single-turnover excision (kst). Normalized rates are shown, obtained by dividing the rate constant by the maximal rate constant for that substrate. Measurements are the average of ≥3 independent determinations, and the S.D. is ≤ 15% of the value. Unless otherwise indicated, the reaction conditions were 50 mm sodium acetate, pH 6.0, 100 mm ionic strength, 0.1 mg/ml bovine serum album in, 1 mm dithiothreitol, 1 mm EDTA, and 37 °C.GA7mGb22 °C, sodium HEPES, pH 7.7.ϵAPaired with: Thymine0.220.00230.980.68 Cytosine0.00400.0910.020.54 Guanine0.0810.0120.460.22 Adenine(1)(1)(1)(1)a For single-turnover excision (kst). Normalized rates are shown, obtained by dividing the rate constant by the maximal rate constant for that substrate. Measurements are the average of ≥3 independent determinations, and the S.D. is ≤ 15% of the value. Unless otherwise indicated, the reaction conditions were 50 mm sodium acetate, pH 6.0, 100 mm ionic strength, 0.1 mg/ml bovine serum album in, 1 mm dithiothreitol, 1 mm EDTA, and 37 °C.b 22 °C, sodium HEPES, pH 7.7. Open table in a new tab 1,N6-Ethenoadenosine is a bulky alkylated adduct of adenosine that does not stably pair with thymidine, providing an opportunity to further test the origin of the opposing base effects observed for 7mG. The AlkA-catalyzed excision of 1,N6-ethenoadenine (ϵA) from DNA showed little dependence upon the identity of the opposing base. Less than 2-fold differences were observed for excision of ϵA when it was paired opposite from A, T, or C (Table I), suggesting that AlkA does not have a strong preference for the identity of the opposing base. This is consistent with the absence of specific protein contacts to the opposing base in the crystal structure of AlkA bound to a DNA inhibitor (3Hollis T. Ichikawa Y. Ellenberger T. EMBO J. 2000; 19: 758-766Crossref PubMed Scopus (206) Google Scholar). The observed 5-fold decrease in the rate constant for excision of ϵA from an ϵA·G pair relative to an ϵA·A pair (Table I) is consistent with the favorable base pairing interactions of an ϵA·G base pair observed in the crystal structure of an ϵA-containing oligonucleotide duplex (25Biswas T. Clos L.J. SantaLucia J. Mitra S. Roy R. J. Mol. Biol. 2002; 320: 503-513Crossref PubMed Scopus (25) Google Scholar, 28Leonard G.A. McAuley-Hecht K.E. Gibson N.J. Brown T. Watson W.P. Hunter W.N. Biochemistry. 1994; 33: 4755-4761Crossref PubMed Scopus (38) Google Scholar). The absence of strong opposing base effects in the excision of ϵA and the inverse correlation between base pair stability and excision rate for 7mG and for normal purines (see below) suggest that the base flipping step serves as a barrier to the excision of normal bases present in Watson-Crick base pairs. AlkA-catalyzed Excision of Normal Bases—AlkA shows low levels of activity toward all four of the normal DNA bases in genomic DNA with the greatest activity toward G and A (15Berdal K.G. Johansen R.F. Seeberg E. EMBO J. 1998; 17: 363-367Crossref PubMed Scopus (156) Google Scholar). As 7mG is more efficiently excised from a mismatch (Table I), we surmised that AlkA might excise normal bases more efficiently if they reside in mismatched base pairs. This possibility was tested by measuring the AlkA-catalyzed excision of G and A with different opposing bases. Both G and A are preferentially excised from mismatched base pairs in a defined oligonucleotide sequence (Fig. 3). As much as 400-fold greater glycosylase activity was observed for excision of mismatched purines relative to purines in Watson-Crick base pairs (Table I). Surprisingly, additional sites of AlkA-catalyzed base excision were detected (Fig. 3). Notably purines were excised from an A·T base pair and the neighboring G·C base pair near the end of the oligonucleotide with rate constants significantly larger than those observed for the excision of either G·C or A·T present at the central position of this 25-mer oligonucleotide (Fig. 3 and data not shown). This preferential excision could be due to the different sequence context or to an end-binding effect. AlkA showed preferential excision of normal purines from sites near the 5′ end of other oligonucleotides that were examined but also excises internal purines when they are present in the sequence (data not shown). This suggests the presence of hot spots in the genome for gratuitous repair that could significantly increase mutation rates at these sites. To confirm that AlkA was responsible for the excision of unmodified bases from mismatched base pairs, we purified an active site mutant (D238N) that has no detectable glycosylase activity toward alkylated bases (Ref. 22Labahn J. Schärer O.D. Long A. Ezaz-Nikpay K. Verdine G.L. Ellenberger T.E. Cell. 1996; 88: 321-329Abstract Full Text Full Text PDF Scopus (232) Google Scholar and data not shown) and tested its activity toward purine-containing mismatches. The D238N mutant does not show detectable glycosylase activity toward mismatch-containing oligonucleotide duplexes, either toward the central mismatch or toward the correctly paired bases near the end of the DNA that were excised by wild-type AlkA (data not shown). This confirms that AlkA, and not another contaminating glycosylase, is responsible for the mismatch-specific base excision that we observe. Undamaged purines are clearly better substrates than undamaged pyrimidines, since excision of normal bases occurs preferentially at purines in a Watson-Crick paired DNA duplex (Fig. 3). However, AlkA can slowly excise normal pyrimidines from PCR-amplified DNA (15Berdal K.G. Johansen R.F. Seeberg E. EMBO J. 1998; 17: 363-367Crossref PubMed Scopus (156) Google Scholar), and some alkylated and oxidized pyrimidines are relatively good substrates (29McCarthy T.V. Karran P. Lindahl T. EMBO J. 1984; 3: 545-550Crossref PubMed Scopus (205) Google Scholar). We did not detect excision of normal pyrimidines from Watson-Crick base pairs using our assay, presumably because the rate constants for excision of normal purines are substantially greater than for excision of normal pyrimidines, and the presence of the resulting apurinic sites inhibits any subsequent binding to and excision of pyrimidine bases. However, AlkA-catalyzed excision was observed for pyrimidine·pyrimidine mismatches (Table II). The single-turnover rate constants for excision of undamaged pyrimidines are only 9% (C·C), 1% (T·C), and 0.1% (U·C) that of the rate constant for excision of G from a G·T mismatch. These results confirm and extend the previous finding that AlkA has significant glycosylase activity toward each of the normal bases in DNA (15Berdal K.G. Johansen R.F. Seeberg E. EMBO J. 1998; 17: 363-367Crossref PubMed Scopus (156) Google Scholar).Table IIRate and equilibrium constants for DNA binding and base excision by AlkASubstratekstaSingle-turnover rate constants for base excision were measured with saturating AlkA. Measurements are the average of ≥3 independent determinations and the S.D. is ≤15% of the value in all cases. Unless otherwise stated, enzymatic reaction conditions are 37 °C, 50 mm sodium acetate, pH 6.0, 1 mm dithiothreitol, 1 mm EDTA, and 0.1 mg/ml bovine serum albumin. The ionic strength was adjusted to 100 mm with NaCl.knonbThe nonenzymatic data are for 37 °C from Ref. 26 unless otherwise indicated.KdcUnless otherwise indicated, the binding constants were determined by fluorescence anisotropy for binding to wild-type AlkA as described under "Experimental Procedures."kst/KdRate enhancementdThe rate enhancement is defined as kst/knon, and the catalytic proficiency is defined as (kcat/Km)/kw in which kw is the bimolecular nonenzymatic rate constant for N-glycosidic bond hydrolysis (kw = knon/55 m).Catalytic proficiencydThe rate enhancement is defined as kst/knon, and the catalytic proficiency is defined as (kcat/Km)/kw in which kw is the bimolecular nonenzymatic rate constant for N-glycosidic bond hydrolysis (kw = knon/55 m).min-1min-1nmm-1s-1×103×10137mG (C)1.21.6 × 10-4230eThe D238N mutant was used to prevent excision of 7mG. The mutant bound to G·C DNA with a Kd value of 230 nm, which is the same within error as the Kd value for wild-type AlkA binding to this DNA (Kd = 280 nm).8.7 × 1041.40.187mG (T)300fThe rate constant was estimated by measuring the rate consta