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Interactions of the human, rat, Saccharomyces cerevisiae and Escherichia coli 3-methyladenine-DNA glycosylases with DNA containing dIMP residues

Interactions of the human, rat, Saccharomyces cerevisiae and Escherichia coli 3-methyladenine-DNA... 1332–1339 Nucleic Acids Research, 2000, Vol. 28, No. 6 © 2000 Oxford University Press Interactions of the human, rat, Saccharomyces cerevisiae and Escherichia coli 3-methyladenine-DNA glycosylases with DNA containing dIMP residues Murat Saparbaev, Jean-Claude Mani and Jacques Laval* Groupe ‘Réparation des lésions Radio- et Chimio-Induites’, UMR 8532 CNRS, Institut Gustave Roussy, 94805 Villejuif Cedex, France and UMR 9921 CNRS, 15, Avenue Charles Flahaut, 34060 Montpellier Cedex, France Received December 9, 1999; Revised and Accepted February 3, 2000 ABSTRACT be important for the understanding of the molecular mecha- nisms involved in DNA repair. In DNA, the deamination of dAMP generates 2′-deoxy- The hydrolytic deamination of dCMP and dAMP residues in inosine 5′-monophosphate (dIMP). Hypoxanthine DNA yields dUMP and dIMP residues, respectively (1,2). In (HX) residues aremutagenic sincetheygiveriseto Escherichia coli, the removal of uracil from DNA is performed A·T→G·C transition. They are excised, although with by the uracil-DNA glycosylase (3) and by the mismatch- different efficiencies, by an activity of the 3-methyl- specific G/U-DNA glycosylase (4) whereas the excision of hypoxanthine (HX) is performed by an activity associated with adenine (3-meAde)-DNA glycosylases from Escherichia the 3-methyladenine (3-meAde)-DNA glycosylase (5). This coli (AlkA protein), human cells (ANPG protein), rat latter activity is associated in human cells with the ANPG cells (APDG protein) and yeast (MAG protein). protein (6,7), in rat cells with the APDG protein (8), in yeast Comparison of the kinetic constants for the excision with the MAG protein (9) and in E.coli with the AlkA protein of HX residues by the four enzymes shows that the coded for by the alkA gene (10). The mutagenic properties of E.coli and yeast enzymes are quite inefficient, dIMP residues have been ascertained by site-specific mutagen- whereasfor the ANPG and theAPDG proteinsthey esis in vivo (11,12). A single dIMP residue, inserted in vitro at a specific locus in a M13mp9 RF molecule, exhibits miscoding repair the HX residues with an efficiency comparable properties leading to mutagenesis in E.coli (11). Furthermore, to that of alkylated bases, which are believed to be in mammalian cells, a synthetic c-Ha-ras gene containing HX the primary substrates of these DNA glycosylases. resulted in A·T→G·C transition (12). Since the use of various substrates to monitor the The presence of hot spots of mutagenesis in E.coli,as well as activity of HX-DNA glycosylases has generated in human cells, increases the possibility that the base context conflicting results, the efficacy of the four 3-meAde-DNA next to the lesion could lower the efficacy of the repair of a glycosylases of different origin was compared using modified base. A significant variation in the rate of repair, depending upon the surrounding sequence context, has been three different substrates. Moreover, using oligo- shown in the case of repair of uracil (13), AP-sites (14), forma- nucleotides containing a single dIMP residue, we midopyrimidine residues (15), O -methylguanine (16), N- investigated a putative sequence specificity of the methylated bases (17) and UV-induced photoproducts (18). enzymes involving the bases next to the HX residue. The slow rate of repair of cyclobutane dimers in the human p53 We found up to 2–5-fold difference in the rates of HX gene at mutational hotspots in skin cancer is documented, excision between the various sequences of the oligo- suggesting that hotspots for mutations arise partly as a conse- nucleotides studied. When the dIMP residue was quence of heterogeneous repair within specific sequences (19). placed opposite to each of the four bases, a preferential The aim of the present study was to investigate the structural requirements for the interaction of the 3-meAde-DNA glyco- recognition of dI:T over dI:dG, dI:dC and dI:dA sylases of different origin with dIMP residues, when present in mismatches was observed for both human (ANPG) different sequence contexts. The best substrate is a double- and E.coli (AlkA) proteins. At variance, the yeast stranded oligonucleotide containing a single HX residue. The MAG protein removed more efficiently HX from a results show that pure preparations of human, rat, yeast and dI:dG over dI:dC, dI:T and dI:dA mismatches. E.coli 3-meAde-DNA glycosylases have dramatically different HX excision abilities according to the sequence context. Up to 2–5-fold difference in the rates of HX removal INTRODUCTION between various sequences was measured. Moreover, the The rules that govern the recognition of a damaged base by the bacterial, yeast and mammalian enzymes show definite enzyme in charge of its elimination, in vitro and in vivo,would sequence preference. *To whom correspondence should be addressed. Tel: +33 1 42114824; Fax: +33 1 42114454; Email: jlaval@igr.fr Nucleic Acids Research, 2000, Vol. 28, No. 6 1333 MATERIALS AND METHODS abasic sites) (29) and limiting amounts of enzyme. In the case of human, rat and yeast 3-meAde-DNA glycosylases, the incu- Bacterial strains and plasmids bation mixture was supplemented with 100 mM KCl. Incuba- Escherichia coli BH290 (X::tagA1, alkA1, thy-hsdR)is a tions were for 30 min at 37°C unless otherwise stated. The derivative of AB1157 (20) and was from laboratory stocks. reaction was stopped by addition of 12 µ lof 3 MNaCl, The pALK10 plasmid (8) is a subclone from pYN1000 (10) followed by extraction with an equal volume of phenol– containing the E.coli alkA gene. The pBKY143 plasmid chloroform (1:1). The samples were centrifuged for 3 min, the containing the yeast mag gene (9), coding for the yeast 3- aqueous phase was collected and the DNA precipitated by methyladenine-DNA glycosylase, was a gift from Dr E. addition of 2 µ l of 0.25% linear polyacrylamide (30) and 300 µ l Seeberg (University of Oslo, Norway). of ethanol (–20°C). The precipitate was recovered by centrifu- gation, dried and dissolved in 10 µ l of formamide, heated for Enzymes 3min at 90°C, loaded onto 20% polyacrylamide gel containing The E.coli Fpg protein was purified as described (21). The 7 M urea and electrophoresed in Tris–Borate–EDTA buffer. AlkA protein prepared as described (22) was a gift from Dr B. The gels were subjected to autoradiography. For quantification, Tudek (this laboratory). The ANPG40 protein, the truncated the gels were exposed to a Storm 840 Phosphor Screen and the 26 kDa, 230 amino acids human enzyme (6) and the APDG amounts of radioactivity in the bands were quantified using the TM protein, the rat enzyme (8) were prepared by G. Chyzak (this ImageQuaNT Software. The data given are from a single experi- laboratory). The MAG protein was purified as described (23). ment, but replicates were consistently within 5%. 3-meAde-DNA glycosylase standard assays were performed in the incubation Materials mixture described above to determine the HX-DNA glycosylase Nucleic acids and nucleotides were purchased from Roche 3 activity, but using as substrate [ H]DMS-DNA and measuring (Mannheim, Germany). Radiolabeled reagents were obtained the ethanol-soluble radioactive products (6–8,25). from the following sources: [ H]dimethyl-sulfate (DMS) (3.8 Ci/mmol) from New England Nuclear, [γ- P]dATP Table 1. Sequences of duplex oligonucleotides used for measuring the (3000 Ci mmol) and [1′,2′,2,8,- H]dATP (92 Ci/mmol) from HX-DNA glycosylase activity Amersham-Pharmacia. [ H]dITP was prepared by deamination of [ H]dATP with nitrous acid and the products were purified and analyzed as described (24). Substrates The preparation of [ H]dIMP double-stranded DNA (160 000 c.p.m./µ g) was as described (5) and had a specific activity of 51 c.p.m./fmol of dIMP residue. This substrate will hereafter be referred to as [ H]dIMP-M13mp8 DNA. Poly(dGC-dGC) containing [ H]dIMP residues (80 000 c.p.m./µ g) was prepared as described for the preparation of [ H]dIMP- M13mp8 substrate but using poly(dGC-dGC). Its specific activity was 51 c.p.m./fmol of dIMP residue. Alkylated DNA, I, deoxyinosine residue; N, one of the four natural deoxynucleosides T, 3 3 dA, dG and dC, respectively. [ H]DMS-DNA, was prepared as described using [ H]DMS 3 Sequence used by Dianov and Lindahl (32). (25,26). The specific activities of the H-methylated substrates were 1995 c.p.m./pmol of methylated bases. Radioactively labeled double-stranded oligonucleotides HPLC chromatography Single-stranded oligonucleotides containing or without dIMP HPLC chromatography was performed as already described residues were synthesized by Dr E. Lescot (this laboratory) or (5) to isolate and subsequently measure [ H]HX after reaction purchased from Genset (Paris, France). The sequence of 3 3 of the enzyme with [ H]dIMP-M13mp8 DNA or [ H]dIMP- duplex oligonucleotides that we chose was part of the gpt gene poly(dGC–dGC). (27). The sequences of the various oligonucleotides used are shown in Table 1. Surface plasmon resonance analysis of the interaction Aliquots of 30 pmol of single-stranded oligonucleotide were between 3-meAde-DNA glycosylase and the immobilized 5′-end labeled with [γ- P]ATP then annealed with a non- oligonucleotide HX20-34/T radioactive complementary oligonucleotide, as described (5). Kinetics of interactions were determined using BIAcore tech- The formation of double-stranded oligonucleotides was moni- nology (BIAcore AB, Uppsala, Sweden) which allows real- tored by 15% PAGE under non-denaturing conditions (28). time analysis of unlabeled compounds. Biotinylated oligo- DNA glycosylase assays nucleotide HX20-34/T (5 µ g/ml in HBS buffer containing 10 mM HEPES–KOH pH 7.4, 150 mM NaCl, 3.4 mM EDTA, HX-DNA glycosylase standard reaction mixture (100 µ l) 0.005% BIAcore surfactant P20) was injected at a flow rate of contained 1 pmol of P-5′-end labeled oligonucleotide duplex, 50 mM HEPES–KOH pH 7.8, 1 mM EDTA, 5 mM β-mercapto- 5 µ l/min onto a streptavidin coated sensorchip (SA sensor chip, ethanol, 100 µ g/ml BSA (Molecular Biology Grade, Roche BIAcore). The amount of immobilized oligonucleotide was found Mannheim), 20 U E.coli Fpg protein (in order to incise DNA at to be ~30–50 pg/mm (30–50 RU). Different concentrations of the 1334 Nucleic Acids Research, 2000, Vol. 28, No. 6 (lane 3), treatments that will reveal AP sites, it is not incised, showing that it does not contain the AP site. When this double- stranded oligodeoxyribonucleotide is treated either with the ANPG40 protein (lane 4) or the AlkA protein (lane 10), it is only minimally incised due to the instability of the generated abasic site under the experimental conditions used. However, when the duplex DNA is treated first by the ANPG40 protein (lanes 5–8) or by the AlkA protein (lanes 10–14) then by the FPG (lanes5 and11),the Nfo(lanes7and13),the Nth(lanes 8 and 14) proteins or with hot piperidine (lanes 6 and 12), ~70% of the 34mer oligonucleotide are converted to a product migrating at the position of the 19mer. This is due to the excision of HX residue followed by the incision of the oligo- Figure 1. Activity of the ANPG40 and AlkA proteins on duplex deoxyribo- nucleotide at the AP site according either to a β–δ elimination nucleotides containing dIMP residues. The P-5′-end labeled double-stranded mechanism (lanes 5, 6, 11 and 12), or a hydrolytic mechanism oligonucleotide HX20-34/T, after various treatments, was analyzed by PAGE (lanes 7 and 13), or a β-elimination mechanism (lanes 8 and under denaturing conditions. Lane 1, no treatment; lane 2, treated with piperidine 14) (31). It should be noted that both proteins have an absolute (1 M, 30 min, 90°C); lane 3, treated with the Fpg protein (100 ng, 20 min, 37°C); lanes 4–8, HX20-34/T was treated with the ANPG40 protein (0.5 U, 20 min, requirement for a duplex oligonucleotide, since no detectable 37°C); lanes 10–14, treated with the AlkA protein (5 U, 60 min, 37°C). These activity was observed when the ANPG40 (lane 9) or the AlkA samples were then further treated (lanes 5 and 11) either with piperidine (1 M, proteins (data not shown) act on a single-stranded oligonucleo- 30 min, 90°C) or with abasic site nicking enzymes (30 min at 37°C). Lanes 6 and tide containing a dIMP residue. In conclusion, both the 12, with the Fpg protein (100 ng); lanes 7 and 13, with the Nfo protein (500 ng); lanes 8 and 14, with the Nth protein (100 ng). Lane 9, the P-5′-end labeled single- ANPG40 and the AlkA proteins act upon DNA containing stranded oligonucleotide HX20-34/T was treated with the ANPG40 and Fpg dIMP residues as a DNA glycosylase leaving an AP site. This proteins as above. Arrow a indicates the 34mer oligonucleotide. Arrow b also holds true for the rat APDG and the yeast MAG proteins points to the 19mer oligonucleotide with an α–β unsaturated aldehyde at the (data not shown). 3′-terminus. Arrow c points to the 19mer oligonucleotide with 3′-OH group at the 3′-terminus. Arrow d points to the 19mer oligonucleotide with a phosphate Comparison of the various substrates used to monitor the group at the 3′-terminus. For details see Materials and Methods. HX-DNA glycosylase activity and kinetic constants of the various 3-meAde-DNA glycosylases for substrates enzymes (ANPG40 and AlkA) were then injected at a flow rate containing dIMP residues of 10 µ l/min and at the end of the association time (300 s), Various substrates have been used, in different laboratories, to dissociation occurred by adding running buffer (HBS) for 300 evaluate the HX-DNA glycosylase activity, including DNA of s. The kinetic parameters of the binding reactions were deter- M13mp8 or poly(dGC–dGC) containing [ H]dIMP residues mined using BIAevaluation 2.1 software (BIAcore). and a duplex oligodeoxynucleotide containing a unique dIMP residue at a precise location. These substrates were used in order to compare and to ascertain their relative properties. As RESULTS shown in Table 2, the duplex oligonucleotide containing a Human ANPG, rat APDG, yeast MAG and E.coli AlkA dIMP residue is by far the best substrate for all the three proteins excise HX residues from duplex enzymes. This observation leads to the use of this latter oligodeoxyribonucleotide containing dIMP substrate to establish the kinetic parameters and the influence of the sequence context upon the excision of HX by the various As shown in Figure 1 the double-stranded oligodeoxyribo- DNA repair proteins. nucleotide containing a dIMP residue at position 20 migrates as a single species at position 34mer (lane 1). When it is treated The results presented in Figure 2A show the time course either with piperidine (lane 2) or by an excess of Fpg protein release of the HX residues from the duplex oligonucleotide Table 2. Comparison of the activity of 3-meAde-DNA glycosylases of various origin on [ H]dIMP M13mp8 DNA, [ H]dIMP poly(dGC–dGC) or duplex oligonucleotide HX20-34/T 34mer Substrate Hypoxanthine released (fmol/min) by 1 U of ANPG40 protein MAG protein AlkA protein 3 b –3 –3 [ H]dIMP M13mp8 DNA 1.05 ~0.1 × 10 5 × 10 3 b –3 –3 [ H]dIMP poly(dGC–dGC) 0.5 ~0.1 × 10 12 × 10 c d d d Oligonucleotide containing a single dIMP residue 311 0.4 1.2 One unit of 3-meAde-DNA glycosylase (of any origin) is the amount of protein liberating 1.0 pmol of methylated base as ethanol-soluble products from [ H]DMS-DNA, in 1 min at 37°C. b 3 After incubation of the various substrates containing [ H]dIMP residues (2 nM) with the respective enzyme, the products of the reaction were separated by HPLC and the fractions analyzed for radioactivity. For details see Materials and Methods. Substrate is HX20-34/T and the concentration of dIMP residues is 10 nM. Data taken from (5). Nucleic Acids Research, 2000, Vol. 28, No. 6 1335 containing a dIMP residue treated by the ANPG40 protein. It is measured by the appearance of a new band, migrating at the position of the 19mer, generated by the β-lyase activity of the Fpg protein at the AP site produced by the DNA glycosylase activity. The quantification of the amount of radioactivity migrating at the position of the 19mer shows that it increased linearly during the initial part of the reaction (Fig. 2B). Similar kinetics were obtained with the other proteins and allowed the determination of the initial velocities of the enzymatic reac- tions used to determine the kinetic constants. The DNA glyco- sylase kinetics constants of the ANPG40, the MAG and the AlkA proteins, using HX20-34/T oligodeoxynucleotide as substrate, are listed in Table 3. These data show a dramatic difference between the three enzymes, the human one being by far the most efficient. For comparison purposes, it should be recalled that the K for excision of 3-meAde by the ANPG –1 protein is 8 nM, the turnover number k =11 min , the specif- cat –1 –1 icity constant k /K =1.4 nM min and for the excision of cat m –1 the 7-MeGua, K =25 nM, k =0.35 min and k /K =0.014 m cat cat m –1 –1 nM min (7). Therefore the k /K valuefor theexcision of cat m HX present in the HX20-34/T oligodeoxynucleotide shows that the specificity of the reaction is comparable to the release of 7-meGua residues but it is less efficient than for the release Figure 2. Kinetics of excision, by the ANPG40 protein, of HX from an oligo- nucleotide duplex containing thymine opposite a deoxyinosine residue. of 3-meAde residues. (A) The oligonucleotide HX20-34/T (10 nM) was incubated with the ANPG40 protein (0.2 U) for increasing periods of time in the presence of Fpg protein. The products of the reaction were separated by electrophoresis on a 20% poly- Table 3. Kinetic constants for the ANPG40, MAG and AlkA proteins for acrylamide gel containing 7 M urea and visualized. Lanes 1–7, incubation time excision of HX from duplex oligonucleotide containing dIMP residue 0, 5, 10, 15, 20, 25 and 30 min, respectively. (B) The radioactivity of the products of the reaction shown in (A) was quantified and plotted as a function a –1 b Protein K (nM) k (min ) k /K K (nM) m cat cat m D of time. For details see Materials and Methods. –2 –3 ANPG40 6 21 × 10 35 × 10 23.5 –3 –6 MAG ~1000 ~15 × 10 ~15 × 10 nd –3 –6 AlkA 420 0.89 × 10 2 × 10 232 protruding ends and has different 3′ and 5′ flanking bases: in this case the efficiency of the ANPG40 protein is only 1.6-fold The apparent K of the 3-meAde-DNA glycosylases of different origin were higher than that observed when using sticky ends (Table 4A). determined by incubating the respective protein with increasing concentrations of HX20-34/T oligonucleotide. The oligonucleotide concentrations range were The sequence context effect was further explored using the 5–40 nM for the ANPG40 protein and 100–1000 nM for the MAG and AlkA oligonucleotides HX20-34/T and HX13-34/T which are proteins. The initial velocities measured for excision of HX were treated as identical but contain the dIMP residue in a different context: described by Lineweaver and Burk. 5′-GpIpC-3′ and 5′-CpIpC-3′, respectively. The latter substrate b 2 30–50 pg/mm of biotinylated HX20-34/T was immobilized onto a streptavidin is recognized 1.5-fold more efficiently. coated sensor chip in a BIAcore. Various concentrations of each enzyme were then injected at a flow rate of 10 µ l/min. The kinetic parameters ka and kd For comparison purpose with the previous investigation of were determined using BIAevaluation 2.1 software (BIAcore) and K calculated Dianov and Lindahl (32) using purified preparations from calf as kd/ka. nd, Not determined. thymus extracts, their a, c, d and e sequences (32) were included and are in this work sequences HX20-DL/T, HX20- Effect of the sequence context and the nature of the base DL/A, HX20-DL/C and HX20-DL/C, respectively. opposite the dIMP residue on the activity of the The potential importance of the sequence context, next to the mammalian ANPG and APDG proteins lesion, was investigated using HX20-DL/T which has sticky ends, the same length, the same location of the dIMP residue in The initial rate of cleavage of various substrates having the duplex oligonucleotide as HX20-40/T but has different different sequence context by the mammalian 3-meAde-DNA overall sequence and lesion context (a dG 5′ to the lesion glycosylases was compared. Moreover, this study was instead of a dC). As shown in Table 4A, the human ANPG40 extended by using, for each oligonucleotide, duplexes protein is the most active when using as substrate HX20-DL/T, containing dC, dG or dA positioned opposite the dIMP residue. in this sequence dIMP residue is flanked by two dCs and the Since it has been shown that the activity of a purified fraction duplex has two sticky ends. The results show a dramatic of calf thymus HX-DNA glycosylase was not affected by the change (5.5-fold) in the efficiency of the repair that could be base composition of the oligomer being either A·T or G·C rich attributed to the sequence context. (32), this study was focused on a random sequence, by using a stretch of a natural gene (27). However, the present data do not give a clear explanation concerning the specific features that make HX20-DL the best The potential importance of sticky as compared to blunt ends was investigated using HX20-34 which is identical to the core substrate for the ANPG40 and APDG proteins. It is clear that part (31 bases) of HX20-40, but does not contain the 5′ the two flanking bases are not the main reason for the good or 1336 Nucleic Acids Research, 2000, Vol. 28, No. 6 Table 4. Initial velocities of cleavage, by various mammalian 3-methyladenine DNA glycosylases, of oligonucleotide duplexes having different structures and containing different bases opposite to the dIMP residue A. The human ANPG40 protein Oligonucleotides HX20-40 HX20-34 HX13-34 HX20-DL Mismatch Hypoxanthine released by 1 U of the ANPG40 protein (fmol/min) dI:T 132 212 316 728 dI:dC 82 111 nd 82 dI:dG 85 128 nd 309 dI:dA 64 68 nd 94 B. The rat APDG protein Oligonucleotides HX20-40 HX20-34 HX13-34 HX20-DL Mismatch Hypoxanthine released by 1 U of the APDG protein (fmol/min) dI:T 85 101 122 141 dI:dC 43 53 nd 61 dI:dG 45 48 nd 123 dI:dA 19 20 nd 53 Sequences from Dianov and Lindahl (32). Different oligonucleotides containing a single dIMP at a defined position in various sequence context were annealed with the complementary oligonucleotides in order to generate the mismatches dI:T, dI:dC, dI:dG and dI:dA, and were used as substrates for the ANPG40 or APDG proteins. Initial velocities were calculated from the linear part of the curve where the amount of HX released is plotted as a function of time. For details see Materials and Methods. nd, Not determined. Table 5. Initial velocities of cleavage by the E.coli AlkA protein of oligonucleotide duplexes containing different bases opposite the dIMP residue Oligonucleotides HX20-40 HX20-34 HX13-34 HX20-DL Mismatch Hypoxanthine released by 1 U of the AlkA protein (fmol/min) dI:T 1.35 1.19 0.50 1.18 dI:dC 0.92 0.67 nd 0.48 dI:dG 0.83 0.66 nd 0.16 dI:dA 0.68 0.76 nd 0.77 For details see Table 4. poor removal of HX (compare HX20-DL and HX13-34), but it for a given substrate, which is at variance with the mammalian should be noted that the stability of the duplex containing enzymes, although in most cases dI:T mismatch was the best inosine flanked by pyrimidines is lower than when inosine is repaired. Interestingly, the HX20-DL substrate for all the flanked by purines (33).The behavior of the rat enzyme mismatches shows again in the case of the mismatch dI:dG, (Table 4B) shows a similar profile of activity when compared peculiar behavior. It is very poorly recognized. Among the to the human enzyme. The oligonucleotide HX20-DL/T is the substrates tested, the HX20-40 was the best. best substrate followed by HX13-34/T, HX20-34/T and HX20- The results presented in Table 6 show that as already 40/T. However, the rat enzyme had a lower activity on all the observed for the AlkA protein, the MAG protein has no clear substrates tested. preference for any of the substrates tested. However, regard- The analysis of the efficiency of excision of HX when oppo- less of the sequence context, the excision of HX by the Mag site to each of the four different bases by mammalian 3- protein present in a dI:dG mismatch was the most efficient. meAde-DNA glycosylases reveals that dI:T and dI:dG are the Among the substrates tested, the HX13-34 oligonucleotide was best repaired. the best. The distinctive behavior of the yeast 3-meAde-DNA glycosylase compared to the other enzymes tested should be Effect of the sequence context and the nature of the base noted. opposite the dIMP residue on E.coli AlkA protein and Saccharomyces cerevisiae MAG protein activity DISCUSSION In order to compare the properties of 3-meAde-DNA glycosy- lases from E.coli and yeast to those of the mammalian The DNA repair proteins 3-meAde-DNA glycosylases, regard- enzymes, the initial velocity of the reaction was measured less of their origin, have the ability to release a number of when the AlkA and MAG proteins were acting on the alkylbases (7,8,34), cyclic etheno-adducts (23,35,36), adducts substrates described above. The results presented in Table 5 of nitrogen mustards (37) and HX from DNA containing dIMP show that the AlkA protein does not exhibit any clear preference residues (5). Concerning the excision of HX residues, the Nucleic Acids Research, 2000, Vol. 28, No. 6 1337 Table 6. Initial velocities of cleavage by the yeast MAG protein of oligonucleotide duplexes containing a different base opposite the dIMP residue Oligonucleotides HX20-40 HX20-34 HX13-34 HX20-DL Mismatch Hypoxanthine released by 1 U of the MAG protein (fmol/min) dI:T 0.36 0.40 0.53 0.26 dI:dC 0.34 0.42 nd 0.26 dI:dG 0.42 0.47 nd 0.31 dI:dA 0.28 0.30 nd 0.21 For details see Table 4. human enzyme is by far the most efficient and the yeast MAG The comparison of the kinetic constants for the excision of and E.coli AlkA proteins perform this reaction very poorly (5). HX by the three different enzymes shows that the efficiency of repair by E.coli and yeast 3-meAde-DNA glycosylases are The purpose of this study was to investigate in detail the extremely poor. This is in agreement with the previous in vivo factors that influence the efficacy of four 3-meAde-DNA gly- data showing that, in E.coli, the repair of a site-specifically cosylases of different origin for the same substrate. Since the incorporated dIMP residue was not efficient (11). Taken glycosidic bond of akylated bases is quite unstable and does together these results strongly suggest that, in vivo, HX, as a not allow such detailed studies, we chose to study the activity DNA lesion, is not physiologically important for the fast prop- of these 3-meAde-DNA glycosylases on substrates containing agating monocellular organisms, perhaps due to the slow rate HX residues. This study was facilitated by the use of duplex of adenine deamination in duplex DNA (44). Nevertheless, the oligonucleotides containing dIMP residues at a precise loca- difference between the binding constants of E.coli (AlkA) and tion and the availability of data concerning the structure of human (ANPG40) proteins to an oligonucleotide containing a duplex oligonucleotides containing mismatched dIMP residue dIMP residue was only one order of magnitude. This data (38–42). The evidence from crystal structure and NMR studies possibly means that the inefficient repair of dIMP residue in reveals that (i) the HX base can form stable hydrogen-bonded DNA by bacterial and yeast proteins could be due to an mismatches with all four normal bases T, G, A and C, in DNA extremely slow catalysis. and (ii) a duplex oligonucleotide containing mismatches made The interaction of the human truncated ANPG protein with of dIMP residues mainly takes a B-DNA helix conformation oligodeoxyribonucleotides containing either HX or abasic sites and does not show large perturbations at the local level (38– has been investigated (45). The region of direct protein–DNA 42). interactions consists only of one turn of helix. In order to form The influence of the base opposite the dIMP residue upon the a stable complex between the ANPG protein and HX-DNA, excision of HX residues by the various 3-meAde-DNA glyco- the minimal length of the oligonucleotide should be at least sylases was first investigated. Studies of the thermal stability 20 bp (45). However, the crystal structures of the AlkA protein of oligonucleotides containing deoxyinosine have shown that (46,47) and the truncated ANPG protein complexed to a pyrro- the order of the stability is independent of sequence effects, lidine-containing 13 bp duplex oligonucleotide have been and is: dI:dC > dI:dA > dI:dG > dI:T (33,43). The comparison reported (48). AlkA is a globular protein consisting of three of the efficiency of HX excision by the mammalian 3-meAde- equal-sized domains. Its active site is located in a large hydro- DNA glycosylases shows that it correlates with the thermal phobic cleft rich in aromatic residues between domains 2 and 3 stability of the mismatch: the less stable dI:T and dI:dG are (46,47). The ANPG protein consists of a single domain, the those best repaired. The bacterial enzyme also preferentially core of the protein has a curved antiparallel β sheet with a repairs dI:dT mismatch, although repair of dI:dC, dI:dG and protruding β hairpin which intercalates into the minor groove dI:dA base pairs does not correspond to the thermal stability of of DNA, causing the abasic pyrrolidine nucleotide to flip into the mismatch in the duplex DNA. In the case of the MAG the enzyme active site (48). Recognition of damaged bases by protein, the dI:dG base pair was the best repaired. This unusual both proteins involves π-electron stacking interactions repair preference for the yeast 3-meAde-DNA glycosylase between electron-rich aromatic side chains of the protein and makes it different from the 3-meAde-DNA glycosylases of electron-deficient alkylated bases (46,48). Both enzymes other species. It should be recalled that NMR studies of the utilize activated water molecules for nucleophilic attack. dI:dG mismatch in DNA indicate the formation of a Based on the crystal structures, it was proposed that in the dI(syn):dG(anti) base pair in a B-DNA helix (42). As Syn active site of the AlkA protein, the conserved residue Asp238 conformation of dI takes place only in the dI:dG base pair, it (46) and in ANPG protein, the conserved amino acid Glu125, may be the mechanism that explains the preferential repair of deprotonate the water, resulting in a hydroxide nucleophil the dI:dG base pair by the MAG protein. Since the recognition attacks of the C1′ carbon of the nucleotide (48). According to of a relatively short oligodeoxyribonucleotide by a DNA gly- the general acid and general base catalysis, Asp238 of the cosylase could be modified by the termini of such molecules, AlkA protein and Glu125 of the ANPG protein act as a general the activity of the various enzymes was measured by using the base to abstract a proton from bound water. If one assumes that same sequence in the core of the oligonucleotide but having the catalytic mechanism utilized by the ANPG40 and AlkA blunt or sticky ends. The results show that oligonucleotides proteins is similar to that of the uracil-DNA glycosylase for having blunt ends are somehow better recognized. excision of uracil (49), the nucleophilic attack by water may 1338 Nucleic Acids Research, 2000, Vol. 28, No. 6 not be sufficient in itself to displace damaged bases like HX or ACKNOWLEDGEMENTS 1,N -ethenoadenine (εA), and some other factor would be This work has been supported by grants from European required to increase the efficiency of the leaving group of the Communities, Association pour la Recherche sur le Cancer and base. One may speculate that this could be achieved by the Fondation Franco-Norvégienne pour la Recherche Scientifique protonation of the damaged base and that in the active site of et Technique et le Développement Industriel. the ANPG protein, the totally conserved His136 could act as a general acid to protonate the HX or εA residues. This hypoth- REFERENCES esis might explain the difference of kinetic constants for the 1. Lindahl,T. and Nyberg,B. (1974) Biochemistry, 13, 3405–3410. HX repair between bacterial and human enzymes. Possible 2. Karran,P. and Lindahl,T. (1980) Biochemistry, 19, 6005–6011. absence in the active site of the AlkA protein of an amino acid 3. Lindahl,T., Ljungquist,S., Siegert,W., Nyberg,B. and Sperens,B. (1977) residue which could act as a general acid to protonate HX may J. Biol. Chem., 252, 3286–3294. explain the slow rate of catalysis observed. Since the kinetic 4. Gallinari,P. and Jiriçny,J. (1996) Nature, 383, 735–738. 5. Saparbaev,M. and Laval,J. (1994) Proc. Natl Acad. Sci. USA, 91, constants for the removal of alkylated bases by the AlkA and 5873–5877. ANPG proteins are quite similar (7,50), this could be explained 6. O’Connor,T.R. and Laval,J. (1991) Biochem. Biophys. Res. Commun., by the fact that alkylated bases have in common a positive 176, 1170–1177. 7. O’Connor,T.R. (1994) Nucleic Acids Res., 21, 5561–5569. charge and a labilized glycosidic bond. Therefore one can 8. O’Connor,T.R. and Laval,F. (1990) EMBO J., 9, 3337–3342. tentatively propose that for excision of alkylated bases such as 9. Berdal,K.G., Bjoras,M., Bjelland,S. and Seeberg,E. (1990) EMBO J., 9, 3meAde and 7meGua, nucleophilic attack by water activated 4563–4568. by the general base could be sufficient for catalysis. If it is true, 10. Nakabeppu,Y., Miyala,T., Kondo,H., Iwanaga,S. and Sekiguchi,M. (1984) J. Biol. Chem., 259, 13730–13736. the lower k /K value for the repair of HX and εA residues by cat m 11. Hill-Perkins,M., Jones,M.D. and Karran,P. (1986) Mutat. Res., 162, the AlkA protein could be explained if the active site of the 153–163. AlkA protein lacks the general acid capable to protonate the 12. Kamiya,H., Miura,H., Kato,H., Nishimura,S., Ohtsuka,E. (1992) Cancer Res., 52, 1836–1839. base. 13. Nilsen,H., Yazdankhah,S.P., Eftedal,I. and Krokan,H.E. (1995) FEBS The second aim of the present investigation was to evaluate Lett., 362, 205–209. the influence of the sequence context, namely the nucleotide 14. Haukanes,B.I., Helland,D.E. and Kleppe,K. (1989) Nucleic Acids Res., 17, 1493–1509. sequence 3′ and 5′ next to the dIMP residue, upon its excision 15. Lagravere,C., Malfoy,B., Leng,M. and Laval,J. (1984) Nature, 310, by the various 3-meAde-DNA glycosylases. The results show 798–800. that the bacterial, yeast and mammalian enzymes investigated 16. Georgiadis,P., Smith,C.A. and Swann,P.F. (1991) Cancer Res., 51, have different sequence preferences. The more efficient 5843–5850. 17. Ye,N., Holmquist,G.P. and O’Connor,T.R. (1998) J. Mol. Biol., 284, removal of HX from the HX20-DL/T oligonucleotide was 269–285. performed by the mammalian proteins, whereas the HX20-40/T 18. Brash,D.E., Seetharam,S., Kraemer,K.H., Seidman,M.M. and was preferred by the E.coli AlkA and the HX20-34/G by the Bredberg,A. (1987) Proc. Natl Acad. Sci. USA, 84, 3782–3786. yeast MAG proteins. Since the ANPG40 and APDG proteins 19. Tornaletti,S. and Pfeifer,G.P. (1994) Science, 263, 1436–1438. 20. Boiteux,S., Huisman,O. and Laval,J. (1984) EMBO J., 3, 2569–2573. show, as described above, a clear preference for less thermo- 21. Boiteux,S., O’Connor,T.R., Lederer,F., Gouyette,A. and Laval,J. (1990) dynamically stable mismatches, one can speculate that HX20- J. Biol. Chem., 265, 3916–3922. DL/T has a lower T than the others. Neighboring groups can 22. Tudek,B., Van Zeeland,A.A., Kusmierek,J.T. and Laval,J. (1998) Mutat. Res., 407, 169–176. have a large effect on thermal stability of duplex DNA 23. Saparbaev,M., Kleibl,K. and Laval,J. (1995) Nucleic Acids Res., 23, containing dIMP residues (33), therefore affecting the repair of 3750–3755. the lesion. The peculiar properties of the HX20-DL/N oligo- 24. Karran,P. (1981) In Friedberg,E.C. and Hanawalt,P.C. (eds), DNA Repair: nucleotides, when substrates for the mammalian and E.coli A Laboratory Manual of Research Procedures,Vol. 1.Marcel Dekker, New York, pp. 265–273. enzymes, might be explained by the larger differences in T 25. Laval,J. (1977) Nature, 269, 829–833. values between mismatches, as compared with other oligo- 26. Graves,R.J., Felzenswalb,I., Laval,J. and O’Connor,T.R. (1992) J. Biol. nucleotides. The thermal stability and NMR studies of the Chem., 267, 14429–14435. 27. Palombo,F., Kohfeldt,E., Calcagnile,A., Nehls,P. and Dogliotti,E. (1992) oligonucleotides used can give insight into consensus J. Mol. Biol., 223, 587–594. sequences for good and poor repair of HX from duplex DNA. 28. Castaing,B., Geiger,A., Seliger,H., Nehls,P., Laval,J., Zelwer,C. and However, the sequence effect on the repair of HX was studied Boiteux,S. (1993) Nucleic Acids Res., 21, 2899–2905. in vitro on naked DNA, and it is possible that repair in vivo on 29. O’Connor,T.R. and Laval,J. (1989) Proc. Natl Acad. Sci. USA, 86, 5222–5226. coated DNA could be different. 30. Gaillard,C. and Strauss,F. (1990) Nucleic Acids Res., 18, 378. The generation of 3-meAde-DNA glycosylase deficient 31. Bailly,V., Verly,W.G., O’Connor,T. and Laval,J. (1989) Biochem. J., 262, (APDG ko) mice (51,52) will allow the assessment of the role 581–589. 32. Dianov,G. and Lindahl,T. (1991) Nucleic Acids Res., 19, 3829–3833. of this enzyme in the repair of HX and the physiological rele- 33. Martin,F.H., Castro,M.M., Aboul-ela,F. and Tinoco,I.,Jr (1985) Nucleic vance of such damage in vivo. Unexpectedly, these APDG ko Acids Res., 13, 8927–8938. mice do not exhibit any particular sensitivity to alkylating 34. Nakabeppu,Y., Kondo,H. and Sekiguchi,M. (1984) J. Biol. Chem., 259, 13723–13729. agents (53). Therefore, it will be interesting to test if such 35. Oesch,F., Adler,S., Rettelbach,R. and Doerjer,G. (1986) In Singer,B. and animals will be sensitive to agents generating HX or εA, since Bartsch,H. (eds), The Role of Cyclic and Nucleic Acid Adducts in these two modified bases are excised in vitro with high effi- Carcinogenesis and Mutagenesis, IARC Scientific Publications no. 70. ciency by the APDG protein (5,23). Oxford University Press, New York, NY, pp. 373–379. Nucleic Acids Research, 2000, Vol. 28, No. 6 1339 36. Singer,B., Antoccia,A., Basu,A.K., Dosanjh,M.K., Fraenkel-Conrat,H., 46. Labahn,J., Scharer,O.D., Long,A., Ezaz-Nikpay,K., Verdine,G.L. and Gallagher,P.E., Kusmierek,J.T., Qiu,Z.H. and Rydberg,B. (1992) Proc. Ellenberger,T.E. (1996) Cell, 86, 321–329. Natl Acad.Sci.USA, 89, 9386–9390. 47. Yamagata,Y., Kato,M., Odawara,K., Tokuno,Y., Nakashima,Y., 37. Mattes,W.B., Lee,C.S., Laval,J. and O’Connor,T.R. (1996) Matsushima,N., Yasumura,K., Tomita,K., Ihara,K., Fujii,Y., Carcinogenesis, 17, 643–648. Nakabeppu,Y., Sekiguchi,M. and Fujii,S. (1996) Cell, 86, 311–319. 38. Uesugi,S., Oda,Y., Ikehara,M., Kawase,Y. and Ohtsuka,E. (1987) J. Biol. 48. 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Case-Green,S.C. and Southern,E.M. (1994) Nucleic Acids Res., 22, (1997) Proc. Natl Acad. Sci. USA, 94, 13087–13092. 131–136. 53. Elder,R.H., Jansen,J.G., Weeks,R.J., Willington,M.A., Deans,B., 44. Lindahl,T. (1993) Nature, 362, 709–714. Watson,A.J., Mynett,K.J., Bailey,J.A., Cooper,D.P., Rafferty,J.A, 45. Miao,F., Bouziane,M. and O’Connor,T.R. (1998) Nucleic Acids Res., 26, Heeran,M.C., Wijnhoven,S.W., van Zeeland,A.A. and Margison,G.P. 4034–4041. (1998) Mol. Cell Biol., 18, 5828–5837. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Nucleic Acids Research Oxford University Press

Interactions of the human, rat, Saccharomyces cerevisiae and Escherichia coli 3-methyladenine-DNA glycosylases with DNA containing dIMP residues

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Abstract

1332–1339 Nucleic Acids Research, 2000, Vol. 28, No. 6 © 2000 Oxford University Press Interactions of the human, rat, Saccharomyces cerevisiae and Escherichia coli 3-methyladenine-DNA glycosylases with DNA containing dIMP residues Murat Saparbaev, Jean-Claude Mani and Jacques Laval* Groupe ‘Réparation des lésions Radio- et Chimio-Induites’, UMR 8532 CNRS, Institut Gustave Roussy, 94805 Villejuif Cedex, France and UMR 9921 CNRS, 15, Avenue Charles Flahaut, 34060 Montpellier Cedex, France Received December 9, 1999; Revised and Accepted February 3, 2000 ABSTRACT be important for the understanding of the molecular mecha- nisms involved in DNA repair. In DNA, the deamination of dAMP generates 2′-deoxy- The hydrolytic deamination of dCMP and dAMP residues in inosine 5′-monophosphate (dIMP). Hypoxanthine DNA yields dUMP and dIMP residues, respectively (1,2). In (HX) residues aremutagenic sincetheygiveriseto Escherichia coli, the removal of uracil from DNA is performed A·T→G·C transition. They are excised, although with by the uracil-DNA glycosylase (3) and by the mismatch- different efficiencies, by an activity of the 3-methyl- specific G/U-DNA glycosylase (4) whereas the excision of hypoxanthine (HX) is performed by an activity associated with adenine (3-meAde)-DNA glycosylases from Escherichia the 3-methyladenine (3-meAde)-DNA glycosylase (5). This coli (AlkA protein), human cells (ANPG protein), rat latter activity is associated in human cells with the ANPG cells (APDG protein) and yeast (MAG protein). protein (6,7), in rat cells with the APDG protein (8), in yeast Comparison of the kinetic constants for the excision with the MAG protein (9) and in E.coli with the AlkA protein of HX residues by the four enzymes shows that the coded for by the alkA gene (10). The mutagenic properties of E.coli and yeast enzymes are quite inefficient, dIMP residues have been ascertained by site-specific mutagen- whereasfor the ANPG and theAPDG proteinsthey esis in vivo (11,12). A single dIMP residue, inserted in vitro at a specific locus in a M13mp9 RF molecule, exhibits miscoding repair the HX residues with an efficiency comparable properties leading to mutagenesis in E.coli (11). Furthermore, to that of alkylated bases, which are believed to be in mammalian cells, a synthetic c-Ha-ras gene containing HX the primary substrates of these DNA glycosylases. resulted in A·T→G·C transition (12). Since the use of various substrates to monitor the The presence of hot spots of mutagenesis in E.coli,as well as activity of HX-DNA glycosylases has generated in human cells, increases the possibility that the base context conflicting results, the efficacy of the four 3-meAde-DNA next to the lesion could lower the efficacy of the repair of a glycosylases of different origin was compared using modified base. A significant variation in the rate of repair, depending upon the surrounding sequence context, has been three different substrates. Moreover, using oligo- shown in the case of repair of uracil (13), AP-sites (14), forma- nucleotides containing a single dIMP residue, we midopyrimidine residues (15), O -methylguanine (16), N- investigated a putative sequence specificity of the methylated bases (17) and UV-induced photoproducts (18). enzymes involving the bases next to the HX residue. The slow rate of repair of cyclobutane dimers in the human p53 We found up to 2–5-fold difference in the rates of HX gene at mutational hotspots in skin cancer is documented, excision between the various sequences of the oligo- suggesting that hotspots for mutations arise partly as a conse- nucleotides studied. When the dIMP residue was quence of heterogeneous repair within specific sequences (19). placed opposite to each of the four bases, a preferential The aim of the present study was to investigate the structural requirements for the interaction of the 3-meAde-DNA glyco- recognition of dI:T over dI:dG, dI:dC and dI:dA sylases of different origin with dIMP residues, when present in mismatches was observed for both human (ANPG) different sequence contexts. The best substrate is a double- and E.coli (AlkA) proteins. At variance, the yeast stranded oligonucleotide containing a single HX residue. The MAG protein removed more efficiently HX from a results show that pure preparations of human, rat, yeast and dI:dG over dI:dC, dI:T and dI:dA mismatches. E.coli 3-meAde-DNA glycosylases have dramatically different HX excision abilities according to the sequence context. Up to 2–5-fold difference in the rates of HX removal INTRODUCTION between various sequences was measured. Moreover, the The rules that govern the recognition of a damaged base by the bacterial, yeast and mammalian enzymes show definite enzyme in charge of its elimination, in vitro and in vivo,would sequence preference. *To whom correspondence should be addressed. Tel: +33 1 42114824; Fax: +33 1 42114454; Email: jlaval@igr.fr Nucleic Acids Research, 2000, Vol. 28, No. 6 1333 MATERIALS AND METHODS abasic sites) (29) and limiting amounts of enzyme. In the case of human, rat and yeast 3-meAde-DNA glycosylases, the incu- Bacterial strains and plasmids bation mixture was supplemented with 100 mM KCl. Incuba- Escherichia coli BH290 (X::tagA1, alkA1, thy-hsdR)is a tions were for 30 min at 37°C unless otherwise stated. The derivative of AB1157 (20) and was from laboratory stocks. reaction was stopped by addition of 12 µ lof 3 MNaCl, The pALK10 plasmid (8) is a subclone from pYN1000 (10) followed by extraction with an equal volume of phenol– containing the E.coli alkA gene. The pBKY143 plasmid chloroform (1:1). The samples were centrifuged for 3 min, the containing the yeast mag gene (9), coding for the yeast 3- aqueous phase was collected and the DNA precipitated by methyladenine-DNA glycosylase, was a gift from Dr E. addition of 2 µ l of 0.25% linear polyacrylamide (30) and 300 µ l Seeberg (University of Oslo, Norway). of ethanol (–20°C). The precipitate was recovered by centrifu- gation, dried and dissolved in 10 µ l of formamide, heated for Enzymes 3min at 90°C, loaded onto 20% polyacrylamide gel containing The E.coli Fpg protein was purified as described (21). The 7 M urea and electrophoresed in Tris–Borate–EDTA buffer. AlkA protein prepared as described (22) was a gift from Dr B. The gels were subjected to autoradiography. For quantification, Tudek (this laboratory). The ANPG40 protein, the truncated the gels were exposed to a Storm 840 Phosphor Screen and the 26 kDa, 230 amino acids human enzyme (6) and the APDG amounts of radioactivity in the bands were quantified using the TM protein, the rat enzyme (8) were prepared by G. Chyzak (this ImageQuaNT Software. The data given are from a single experi- laboratory). The MAG protein was purified as described (23). ment, but replicates were consistently within 5%. 3-meAde-DNA glycosylase standard assays were performed in the incubation Materials mixture described above to determine the HX-DNA glycosylase Nucleic acids and nucleotides were purchased from Roche 3 activity, but using as substrate [ H]DMS-DNA and measuring (Mannheim, Germany). Radiolabeled reagents were obtained the ethanol-soluble radioactive products (6–8,25). from the following sources: [ H]dimethyl-sulfate (DMS) (3.8 Ci/mmol) from New England Nuclear, [γ- P]dATP Table 1. Sequences of duplex oligonucleotides used for measuring the (3000 Ci mmol) and [1′,2′,2,8,- H]dATP (92 Ci/mmol) from HX-DNA glycosylase activity Amersham-Pharmacia. [ H]dITP was prepared by deamination of [ H]dATP with nitrous acid and the products were purified and analyzed as described (24). Substrates The preparation of [ H]dIMP double-stranded DNA (160 000 c.p.m./µ g) was as described (5) and had a specific activity of 51 c.p.m./fmol of dIMP residue. This substrate will hereafter be referred to as [ H]dIMP-M13mp8 DNA. Poly(dGC-dGC) containing [ H]dIMP residues (80 000 c.p.m./µ g) was prepared as described for the preparation of [ H]dIMP- M13mp8 substrate but using poly(dGC-dGC). Its specific activity was 51 c.p.m./fmol of dIMP residue. Alkylated DNA, I, deoxyinosine residue; N, one of the four natural deoxynucleosides T, 3 3 dA, dG and dC, respectively. [ H]DMS-DNA, was prepared as described using [ H]DMS 3 Sequence used by Dianov and Lindahl (32). (25,26). The specific activities of the H-methylated substrates were 1995 c.p.m./pmol of methylated bases. Radioactively labeled double-stranded oligonucleotides HPLC chromatography Single-stranded oligonucleotides containing or without dIMP HPLC chromatography was performed as already described residues were synthesized by Dr E. Lescot (this laboratory) or (5) to isolate and subsequently measure [ H]HX after reaction purchased from Genset (Paris, France). The sequence of 3 3 of the enzyme with [ H]dIMP-M13mp8 DNA or [ H]dIMP- duplex oligonucleotides that we chose was part of the gpt gene poly(dGC–dGC). (27). The sequences of the various oligonucleotides used are shown in Table 1. Surface plasmon resonance analysis of the interaction Aliquots of 30 pmol of single-stranded oligonucleotide were between 3-meAde-DNA glycosylase and the immobilized 5′-end labeled with [γ- P]ATP then annealed with a non- oligonucleotide HX20-34/T radioactive complementary oligonucleotide, as described (5). Kinetics of interactions were determined using BIAcore tech- The formation of double-stranded oligonucleotides was moni- nology (BIAcore AB, Uppsala, Sweden) which allows real- tored by 15% PAGE under non-denaturing conditions (28). time analysis of unlabeled compounds. Biotinylated oligo- DNA glycosylase assays nucleotide HX20-34/T (5 µ g/ml in HBS buffer containing 10 mM HEPES–KOH pH 7.4, 150 mM NaCl, 3.4 mM EDTA, HX-DNA glycosylase standard reaction mixture (100 µ l) 0.005% BIAcore surfactant P20) was injected at a flow rate of contained 1 pmol of P-5′-end labeled oligonucleotide duplex, 50 mM HEPES–KOH pH 7.8, 1 mM EDTA, 5 mM β-mercapto- 5 µ l/min onto a streptavidin coated sensorchip (SA sensor chip, ethanol, 100 µ g/ml BSA (Molecular Biology Grade, Roche BIAcore). The amount of immobilized oligonucleotide was found Mannheim), 20 U E.coli Fpg protein (in order to incise DNA at to be ~30–50 pg/mm (30–50 RU). Different concentrations of the 1334 Nucleic Acids Research, 2000, Vol. 28, No. 6 (lane 3), treatments that will reveal AP sites, it is not incised, showing that it does not contain the AP site. When this double- stranded oligodeoxyribonucleotide is treated either with the ANPG40 protein (lane 4) or the AlkA protein (lane 10), it is only minimally incised due to the instability of the generated abasic site under the experimental conditions used. However, when the duplex DNA is treated first by the ANPG40 protein (lanes 5–8) or by the AlkA protein (lanes 10–14) then by the FPG (lanes5 and11),the Nfo(lanes7and13),the Nth(lanes 8 and 14) proteins or with hot piperidine (lanes 6 and 12), ~70% of the 34mer oligonucleotide are converted to a product migrating at the position of the 19mer. This is due to the excision of HX residue followed by the incision of the oligo- Figure 1. Activity of the ANPG40 and AlkA proteins on duplex deoxyribo- nucleotide at the AP site according either to a β–δ elimination nucleotides containing dIMP residues. The P-5′-end labeled double-stranded mechanism (lanes 5, 6, 11 and 12), or a hydrolytic mechanism oligonucleotide HX20-34/T, after various treatments, was analyzed by PAGE (lanes 7 and 13), or a β-elimination mechanism (lanes 8 and under denaturing conditions. Lane 1, no treatment; lane 2, treated with piperidine 14) (31). It should be noted that both proteins have an absolute (1 M, 30 min, 90°C); lane 3, treated with the Fpg protein (100 ng, 20 min, 37°C); lanes 4–8, HX20-34/T was treated with the ANPG40 protein (0.5 U, 20 min, requirement for a duplex oligonucleotide, since no detectable 37°C); lanes 10–14, treated with the AlkA protein (5 U, 60 min, 37°C). These activity was observed when the ANPG40 (lane 9) or the AlkA samples were then further treated (lanes 5 and 11) either with piperidine (1 M, proteins (data not shown) act on a single-stranded oligonucleo- 30 min, 90°C) or with abasic site nicking enzymes (30 min at 37°C). Lanes 6 and tide containing a dIMP residue. In conclusion, both the 12, with the Fpg protein (100 ng); lanes 7 and 13, with the Nfo protein (500 ng); lanes 8 and 14, with the Nth protein (100 ng). Lane 9, the P-5′-end labeled single- ANPG40 and the AlkA proteins act upon DNA containing stranded oligonucleotide HX20-34/T was treated with the ANPG40 and Fpg dIMP residues as a DNA glycosylase leaving an AP site. This proteins as above. Arrow a indicates the 34mer oligonucleotide. Arrow b also holds true for the rat APDG and the yeast MAG proteins points to the 19mer oligonucleotide with an α–β unsaturated aldehyde at the (data not shown). 3′-terminus. Arrow c points to the 19mer oligonucleotide with 3′-OH group at the 3′-terminus. Arrow d points to the 19mer oligonucleotide with a phosphate Comparison of the various substrates used to monitor the group at the 3′-terminus. For details see Materials and Methods. HX-DNA glycosylase activity and kinetic constants of the various 3-meAde-DNA glycosylases for substrates enzymes (ANPG40 and AlkA) were then injected at a flow rate containing dIMP residues of 10 µ l/min and at the end of the association time (300 s), Various substrates have been used, in different laboratories, to dissociation occurred by adding running buffer (HBS) for 300 evaluate the HX-DNA glycosylase activity, including DNA of s. The kinetic parameters of the binding reactions were deter- M13mp8 or poly(dGC–dGC) containing [ H]dIMP residues mined using BIAevaluation 2.1 software (BIAcore). and a duplex oligodeoxynucleotide containing a unique dIMP residue at a precise location. These substrates were used in order to compare and to ascertain their relative properties. As RESULTS shown in Table 2, the duplex oligonucleotide containing a Human ANPG, rat APDG, yeast MAG and E.coli AlkA dIMP residue is by far the best substrate for all the three proteins excise HX residues from duplex enzymes. This observation leads to the use of this latter oligodeoxyribonucleotide containing dIMP substrate to establish the kinetic parameters and the influence of the sequence context upon the excision of HX by the various As shown in Figure 1 the double-stranded oligodeoxyribo- DNA repair proteins. nucleotide containing a dIMP residue at position 20 migrates as a single species at position 34mer (lane 1). When it is treated The results presented in Figure 2A show the time course either with piperidine (lane 2) or by an excess of Fpg protein release of the HX residues from the duplex oligonucleotide Table 2. Comparison of the activity of 3-meAde-DNA glycosylases of various origin on [ H]dIMP M13mp8 DNA, [ H]dIMP poly(dGC–dGC) or duplex oligonucleotide HX20-34/T 34mer Substrate Hypoxanthine released (fmol/min) by 1 U of ANPG40 protein MAG protein AlkA protein 3 b –3 –3 [ H]dIMP M13mp8 DNA 1.05 ~0.1 × 10 5 × 10 3 b –3 –3 [ H]dIMP poly(dGC–dGC) 0.5 ~0.1 × 10 12 × 10 c d d d Oligonucleotide containing a single dIMP residue 311 0.4 1.2 One unit of 3-meAde-DNA glycosylase (of any origin) is the amount of protein liberating 1.0 pmol of methylated base as ethanol-soluble products from [ H]DMS-DNA, in 1 min at 37°C. b 3 After incubation of the various substrates containing [ H]dIMP residues (2 nM) with the respective enzyme, the products of the reaction were separated by HPLC and the fractions analyzed for radioactivity. For details see Materials and Methods. Substrate is HX20-34/T and the concentration of dIMP residues is 10 nM. Data taken from (5). Nucleic Acids Research, 2000, Vol. 28, No. 6 1335 containing a dIMP residue treated by the ANPG40 protein. It is measured by the appearance of a new band, migrating at the position of the 19mer, generated by the β-lyase activity of the Fpg protein at the AP site produced by the DNA glycosylase activity. The quantification of the amount of radioactivity migrating at the position of the 19mer shows that it increased linearly during the initial part of the reaction (Fig. 2B). Similar kinetics were obtained with the other proteins and allowed the determination of the initial velocities of the enzymatic reac- tions used to determine the kinetic constants. The DNA glyco- sylase kinetics constants of the ANPG40, the MAG and the AlkA proteins, using HX20-34/T oligodeoxynucleotide as substrate, are listed in Table 3. These data show a dramatic difference between the three enzymes, the human one being by far the most efficient. For comparison purposes, it should be recalled that the K for excision of 3-meAde by the ANPG –1 protein is 8 nM, the turnover number k =11 min , the specif- cat –1 –1 icity constant k /K =1.4 nM min and for the excision of cat m –1 the 7-MeGua, K =25 nM, k =0.35 min and k /K =0.014 m cat cat m –1 –1 nM min (7). Therefore the k /K valuefor theexcision of cat m HX present in the HX20-34/T oligodeoxynucleotide shows that the specificity of the reaction is comparable to the release of 7-meGua residues but it is less efficient than for the release Figure 2. Kinetics of excision, by the ANPG40 protein, of HX from an oligo- nucleotide duplex containing thymine opposite a deoxyinosine residue. of 3-meAde residues. (A) The oligonucleotide HX20-34/T (10 nM) was incubated with the ANPG40 protein (0.2 U) for increasing periods of time in the presence of Fpg protein. The products of the reaction were separated by electrophoresis on a 20% poly- Table 3. Kinetic constants for the ANPG40, MAG and AlkA proteins for acrylamide gel containing 7 M urea and visualized. Lanes 1–7, incubation time excision of HX from duplex oligonucleotide containing dIMP residue 0, 5, 10, 15, 20, 25 and 30 min, respectively. (B) The radioactivity of the products of the reaction shown in (A) was quantified and plotted as a function a –1 b Protein K (nM) k (min ) k /K K (nM) m cat cat m D of time. For details see Materials and Methods. –2 –3 ANPG40 6 21 × 10 35 × 10 23.5 –3 –6 MAG ~1000 ~15 × 10 ~15 × 10 nd –3 –6 AlkA 420 0.89 × 10 2 × 10 232 protruding ends and has different 3′ and 5′ flanking bases: in this case the efficiency of the ANPG40 protein is only 1.6-fold The apparent K of the 3-meAde-DNA glycosylases of different origin were higher than that observed when using sticky ends (Table 4A). determined by incubating the respective protein with increasing concentrations of HX20-34/T oligonucleotide. The oligonucleotide concentrations range were The sequence context effect was further explored using the 5–40 nM for the ANPG40 protein and 100–1000 nM for the MAG and AlkA oligonucleotides HX20-34/T and HX13-34/T which are proteins. The initial velocities measured for excision of HX were treated as identical but contain the dIMP residue in a different context: described by Lineweaver and Burk. 5′-GpIpC-3′ and 5′-CpIpC-3′, respectively. The latter substrate b 2 30–50 pg/mm of biotinylated HX20-34/T was immobilized onto a streptavidin is recognized 1.5-fold more efficiently. coated sensor chip in a BIAcore. Various concentrations of each enzyme were then injected at a flow rate of 10 µ l/min. The kinetic parameters ka and kd For comparison purpose with the previous investigation of were determined using BIAevaluation 2.1 software (BIAcore) and K calculated Dianov and Lindahl (32) using purified preparations from calf as kd/ka. nd, Not determined. thymus extracts, their a, c, d and e sequences (32) were included and are in this work sequences HX20-DL/T, HX20- Effect of the sequence context and the nature of the base DL/A, HX20-DL/C and HX20-DL/C, respectively. opposite the dIMP residue on the activity of the The potential importance of the sequence context, next to the mammalian ANPG and APDG proteins lesion, was investigated using HX20-DL/T which has sticky ends, the same length, the same location of the dIMP residue in The initial rate of cleavage of various substrates having the duplex oligonucleotide as HX20-40/T but has different different sequence context by the mammalian 3-meAde-DNA overall sequence and lesion context (a dG 5′ to the lesion glycosylases was compared. Moreover, this study was instead of a dC). As shown in Table 4A, the human ANPG40 extended by using, for each oligonucleotide, duplexes protein is the most active when using as substrate HX20-DL/T, containing dC, dG or dA positioned opposite the dIMP residue. in this sequence dIMP residue is flanked by two dCs and the Since it has been shown that the activity of a purified fraction duplex has two sticky ends. The results show a dramatic of calf thymus HX-DNA glycosylase was not affected by the change (5.5-fold) in the efficiency of the repair that could be base composition of the oligomer being either A·T or G·C rich attributed to the sequence context. (32), this study was focused on a random sequence, by using a stretch of a natural gene (27). However, the present data do not give a clear explanation concerning the specific features that make HX20-DL the best The potential importance of sticky as compared to blunt ends was investigated using HX20-34 which is identical to the core substrate for the ANPG40 and APDG proteins. It is clear that part (31 bases) of HX20-40, but does not contain the 5′ the two flanking bases are not the main reason for the good or 1336 Nucleic Acids Research, 2000, Vol. 28, No. 6 Table 4. Initial velocities of cleavage, by various mammalian 3-methyladenine DNA glycosylases, of oligonucleotide duplexes having different structures and containing different bases opposite to the dIMP residue A. The human ANPG40 protein Oligonucleotides HX20-40 HX20-34 HX13-34 HX20-DL Mismatch Hypoxanthine released by 1 U of the ANPG40 protein (fmol/min) dI:T 132 212 316 728 dI:dC 82 111 nd 82 dI:dG 85 128 nd 309 dI:dA 64 68 nd 94 B. The rat APDG protein Oligonucleotides HX20-40 HX20-34 HX13-34 HX20-DL Mismatch Hypoxanthine released by 1 U of the APDG protein (fmol/min) dI:T 85 101 122 141 dI:dC 43 53 nd 61 dI:dG 45 48 nd 123 dI:dA 19 20 nd 53 Sequences from Dianov and Lindahl (32). Different oligonucleotides containing a single dIMP at a defined position in various sequence context were annealed with the complementary oligonucleotides in order to generate the mismatches dI:T, dI:dC, dI:dG and dI:dA, and were used as substrates for the ANPG40 or APDG proteins. Initial velocities were calculated from the linear part of the curve where the amount of HX released is plotted as a function of time. For details see Materials and Methods. nd, Not determined. Table 5. Initial velocities of cleavage by the E.coli AlkA protein of oligonucleotide duplexes containing different bases opposite the dIMP residue Oligonucleotides HX20-40 HX20-34 HX13-34 HX20-DL Mismatch Hypoxanthine released by 1 U of the AlkA protein (fmol/min) dI:T 1.35 1.19 0.50 1.18 dI:dC 0.92 0.67 nd 0.48 dI:dG 0.83 0.66 nd 0.16 dI:dA 0.68 0.76 nd 0.77 For details see Table 4. poor removal of HX (compare HX20-DL and HX13-34), but it for a given substrate, which is at variance with the mammalian should be noted that the stability of the duplex containing enzymes, although in most cases dI:T mismatch was the best inosine flanked by pyrimidines is lower than when inosine is repaired. Interestingly, the HX20-DL substrate for all the flanked by purines (33).The behavior of the rat enzyme mismatches shows again in the case of the mismatch dI:dG, (Table 4B) shows a similar profile of activity when compared peculiar behavior. It is very poorly recognized. Among the to the human enzyme. The oligonucleotide HX20-DL/T is the substrates tested, the HX20-40 was the best. best substrate followed by HX13-34/T, HX20-34/T and HX20- The results presented in Table 6 show that as already 40/T. However, the rat enzyme had a lower activity on all the observed for the AlkA protein, the MAG protein has no clear substrates tested. preference for any of the substrates tested. However, regard- The analysis of the efficiency of excision of HX when oppo- less of the sequence context, the excision of HX by the Mag site to each of the four different bases by mammalian 3- protein present in a dI:dG mismatch was the most efficient. meAde-DNA glycosylases reveals that dI:T and dI:dG are the Among the substrates tested, the HX13-34 oligonucleotide was best repaired. the best. The distinctive behavior of the yeast 3-meAde-DNA glycosylase compared to the other enzymes tested should be Effect of the sequence context and the nature of the base noted. opposite the dIMP residue on E.coli AlkA protein and Saccharomyces cerevisiae MAG protein activity DISCUSSION In order to compare the properties of 3-meAde-DNA glycosy- lases from E.coli and yeast to those of the mammalian The DNA repair proteins 3-meAde-DNA glycosylases, regard- enzymes, the initial velocity of the reaction was measured less of their origin, have the ability to release a number of when the AlkA and MAG proteins were acting on the alkylbases (7,8,34), cyclic etheno-adducts (23,35,36), adducts substrates described above. The results presented in Table 5 of nitrogen mustards (37) and HX from DNA containing dIMP show that the AlkA protein does not exhibit any clear preference residues (5). Concerning the excision of HX residues, the Nucleic Acids Research, 2000, Vol. 28, No. 6 1337 Table 6. Initial velocities of cleavage by the yeast MAG protein of oligonucleotide duplexes containing a different base opposite the dIMP residue Oligonucleotides HX20-40 HX20-34 HX13-34 HX20-DL Mismatch Hypoxanthine released by 1 U of the MAG protein (fmol/min) dI:T 0.36 0.40 0.53 0.26 dI:dC 0.34 0.42 nd 0.26 dI:dG 0.42 0.47 nd 0.31 dI:dA 0.28 0.30 nd 0.21 For details see Table 4. human enzyme is by far the most efficient and the yeast MAG The comparison of the kinetic constants for the excision of and E.coli AlkA proteins perform this reaction very poorly (5). HX by the three different enzymes shows that the efficiency of repair by E.coli and yeast 3-meAde-DNA glycosylases are The purpose of this study was to investigate in detail the extremely poor. This is in agreement with the previous in vivo factors that influence the efficacy of four 3-meAde-DNA gly- data showing that, in E.coli, the repair of a site-specifically cosylases of different origin for the same substrate. Since the incorporated dIMP residue was not efficient (11). Taken glycosidic bond of akylated bases is quite unstable and does together these results strongly suggest that, in vivo, HX, as a not allow such detailed studies, we chose to study the activity DNA lesion, is not physiologically important for the fast prop- of these 3-meAde-DNA glycosylases on substrates containing agating monocellular organisms, perhaps due to the slow rate HX residues. This study was facilitated by the use of duplex of adenine deamination in duplex DNA (44). Nevertheless, the oligonucleotides containing dIMP residues at a precise loca- difference between the binding constants of E.coli (AlkA) and tion and the availability of data concerning the structure of human (ANPG40) proteins to an oligonucleotide containing a duplex oligonucleotides containing mismatched dIMP residue dIMP residue was only one order of magnitude. This data (38–42). The evidence from crystal structure and NMR studies possibly means that the inefficient repair of dIMP residue in reveals that (i) the HX base can form stable hydrogen-bonded DNA by bacterial and yeast proteins could be due to an mismatches with all four normal bases T, G, A and C, in DNA extremely slow catalysis. and (ii) a duplex oligonucleotide containing mismatches made The interaction of the human truncated ANPG protein with of dIMP residues mainly takes a B-DNA helix conformation oligodeoxyribonucleotides containing either HX or abasic sites and does not show large perturbations at the local level (38– has been investigated (45). The region of direct protein–DNA 42). interactions consists only of one turn of helix. In order to form The influence of the base opposite the dIMP residue upon the a stable complex between the ANPG protein and HX-DNA, excision of HX residues by the various 3-meAde-DNA glyco- the minimal length of the oligonucleotide should be at least sylases was first investigated. Studies of the thermal stability 20 bp (45). However, the crystal structures of the AlkA protein of oligonucleotides containing deoxyinosine have shown that (46,47) and the truncated ANPG protein complexed to a pyrro- the order of the stability is independent of sequence effects, lidine-containing 13 bp duplex oligonucleotide have been and is: dI:dC > dI:dA > dI:dG > dI:T (33,43). The comparison reported (48). AlkA is a globular protein consisting of three of the efficiency of HX excision by the mammalian 3-meAde- equal-sized domains. Its active site is located in a large hydro- DNA glycosylases shows that it correlates with the thermal phobic cleft rich in aromatic residues between domains 2 and 3 stability of the mismatch: the less stable dI:T and dI:dG are (46,47). The ANPG protein consists of a single domain, the those best repaired. The bacterial enzyme also preferentially core of the protein has a curved antiparallel β sheet with a repairs dI:dT mismatch, although repair of dI:dC, dI:dG and protruding β hairpin which intercalates into the minor groove dI:dA base pairs does not correspond to the thermal stability of of DNA, causing the abasic pyrrolidine nucleotide to flip into the mismatch in the duplex DNA. In the case of the MAG the enzyme active site (48). Recognition of damaged bases by protein, the dI:dG base pair was the best repaired. This unusual both proteins involves π-electron stacking interactions repair preference for the yeast 3-meAde-DNA glycosylase between electron-rich aromatic side chains of the protein and makes it different from the 3-meAde-DNA glycosylases of electron-deficient alkylated bases (46,48). Both enzymes other species. It should be recalled that NMR studies of the utilize activated water molecules for nucleophilic attack. dI:dG mismatch in DNA indicate the formation of a Based on the crystal structures, it was proposed that in the dI(syn):dG(anti) base pair in a B-DNA helix (42). As Syn active site of the AlkA protein, the conserved residue Asp238 conformation of dI takes place only in the dI:dG base pair, it (46) and in ANPG protein, the conserved amino acid Glu125, may be the mechanism that explains the preferential repair of deprotonate the water, resulting in a hydroxide nucleophil the dI:dG base pair by the MAG protein. Since the recognition attacks of the C1′ carbon of the nucleotide (48). According to of a relatively short oligodeoxyribonucleotide by a DNA gly- the general acid and general base catalysis, Asp238 of the cosylase could be modified by the termini of such molecules, AlkA protein and Glu125 of the ANPG protein act as a general the activity of the various enzymes was measured by using the base to abstract a proton from bound water. If one assumes that same sequence in the core of the oligonucleotide but having the catalytic mechanism utilized by the ANPG40 and AlkA blunt or sticky ends. The results show that oligonucleotides proteins is similar to that of the uracil-DNA glycosylase for having blunt ends are somehow better recognized. excision of uracil (49), the nucleophilic attack by water may 1338 Nucleic Acids Research, 2000, Vol. 28, No. 6 not be sufficient in itself to displace damaged bases like HX or ACKNOWLEDGEMENTS 1,N -ethenoadenine (εA), and some other factor would be This work has been supported by grants from European required to increase the efficiency of the leaving group of the Communities, Association pour la Recherche sur le Cancer and base. One may speculate that this could be achieved by the Fondation Franco-Norvégienne pour la Recherche Scientifique protonation of the damaged base and that in the active site of et Technique et le Développement Industriel. the ANPG protein, the totally conserved His136 could act as a general acid to protonate the HX or εA residues. 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Published: Mar 15, 2000

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