Proc. Natl. Acad. Sci. USA Vol. 91, pp. 5873-5877, June 1994 Biochemistry Excision of hypoxanthine from DNA containing dIMP residues by the Escherichia coli, yeast, rat, and human alkylpurine DNA glycosylases (mutagenesis/DNA repair/multfunctonal enzyme/abasc site/da tion) MURAT SAPARBAEV AND JACQUES LAVAL* Groupe R6paration des 16sions Radio- et Chimio-Induites, URA 147 Centre National de la Recherche Scientifique, Institut Gustave Roussy, 94805 Villejuif Cedex, France Communicated by Philip C. Hanawalt, February 14, 1994
ABSTRACT The deamination of adenine residues in DNA with the 3-methyladenine (3-MeAde) DNA glycosylase generates hypoxanthine, which is mutagenic since it gives rise (TagII; AlkA protein) coded by alkA (10). The alIkA gene is to an A-T to G-C transition. Hypoxanthine is removed by inducible during the adaptive response (11, 12). Furthermore, hypoxanthine DNA glycosylase activity present in Eschenchia we show that the human ANPG protein (13), the rat APDG cofi and mammalia cells. Using polydeoxyribonucleotides or protein (14), and the yeast MAG protein (15), all having double-stranded synthetic oligonucleotides that contain dIMP 3-MeAde DNA glycosylase activity, also excise hypoxan- residues, we show that this activity in E. coli is associated with thine residues from DNA containing dIMP moieties. the 3-methyladenine DNA glycosylase H coded for by the alA gene. This conclusion is based on the following facts: (s) the two MATERIALS AND METHODS enzymatic activities have the same chromatographic behavior on various supports and they have the same molecular weight, Bacterial Strains and Plasmids. E. coli GC4800 (alkAl), (ii) both are induced during the adaptive response, (iii) a GC4802 (alkAl), and BH290 (X::tagAl alkAl, thyA-hsdR) multicopy plasmid bearing the alkA gene overproduces both derivatives of the strain AB1157 (14, 16) and JM105 were activities, (iv) homogeneous preparation of AlkA has both from laboratory stocks. The strain CC106 was obtained from enzymatic activities, (v) the E. coil akA- mutant does not show J. Miller (University of California, Los Angeles) (17). any detectable hypoxanthine DNA glycosylase activity. Under The pALK10 plasmid (14) is a subclone from pYN1000 (10) the same experimental conditions, but using different sub- containing the E. coli alkA gene. The pBKY143 plasmid strates, the same amount of AIkA protein liberates 1 pmol of containing the yeast MAG gene (15), coding for the yeast 3-methyladenine from alkylated DNA and 1.2 fmol of hypo- 3-MeAde DNA glycosylase, was a gift from E. Seeberg xanthine from dIMP-containing DNA. The Km for the latter (University of Oslo, Norway). substrate is 420 x 10-9 M as compared to 5 x 10-9 M for Enzymes. The E. coli FPG protein has been described (18). alkylated DNA. Hypoxanthine is released as a free base during AlkA protein was a gift of B. Tudek and K. Kleibl (this the reaction. Duplex oligodeoxynucleotides containing hypo- laboratory). The human truncated ANPG40 protein (13) was xanthine positioned opposite T. G, C, and A were cleaved a gift of K. Kleibl (this laboratory). The splicing variant efficiently. ANPG protein, APDG protein, and MAG protein- ANPG60 as well as the full-length human ANPG70 proteins the 3-methyladenine DNA glycosylases of human, rat, and (19, 31) and rat APDG protein (14) were gifts from T. yeast origin, respectively-were also able to release hypoxan- O'Connor (this laboratory). The purification of yeast MAG thine from various DNA substrates containing dIMP residues. protein will be described elsewhere. All the proteins were The mammalian enzyme is by far the most efficient hypoxan- purified from crude extract obtained by overproducing the thine DNA glycosylase of all the enzymes tested. protein in E. coli strain BH290 tag- alkA-, to exclude the possibility of E. coli contamination by alkA. In DNA, hydrolytic deamination of adenine and guanine Materials. Nucleic acids, nucleotides, and purine and py- occurs under physiological conditions, yielding hypoxan- rimidine bases were purchased from Boehringer Mannheim. thine and xanthine, respectively (1, 2). Hypoxanthine in Radiolabeled reagents were obtained from the following DNA is potentially mutagenic since it can pair not only with sources: [3H]dimethyl sulfate (DMS) (3.8 Ci/mmol; 1 Ci = 37 thymine but also with cytosine and therefore would result in GBq) was from New England Nuclear, [y-32P]ATP (3000 A-T to G-C transitions after DNA replication (3, 4). Hypo- Ci/mmol) and [1',2',2,8-3H~dATP (92 Ci/mmol) were from xanthine (Hyp) DNA glycosylase excises hypoxanthine from Amersham, [3H]dITP was prepared by deamination of DNA containing dIMP residues in mammalian cells as well as [3H]dATP with nitrous acid as described (20). in Escherichia coli (5-8). Substrate Preparation. M13mp8 single-stranded circular The potential mutagenic properties of dIMP residues have DNA (9 ,ug) was hybridized with reverse sequence primer been substantiated by site-specific mutagenesis in vivo. A (17-mer) (20 ng) by incubation at 60°C for 10 min. Using this single inosine residue at a specific locus in a M13mp9 template, DNA synthesis was performed in a reaction mix- replicative form molecule constructed in vitro exhibits mis- ture (200 ,ul) containing 50 mM Tris HCl (pH 7.5); 10 mM coding mutagenesis in E. coli (3). In mammalian cells, a MgSO4; unlabeled dATP, dCTP, and dTTP (0.25 mM each); synthetic c-Ha-ras gene containing hypoxanthine resulted in 2 AM 13H]dITP; and 10 units of Klenow enzyme (Boehringer increased focus formation (9). Mannheim). After incubation for 3 hr at 37°C, dGTP was During purification ofthe Hyp DNA glycosylase, we found added up to 0.25 mM, followed by incubation for 1 hr at 25°C that in E. coli Hyp DNA glycosylase activity was associated to complete the synthesis. The modified M13 DNA was
The publication costs of this article were defrayed in part by page charge Abbreviations: DMS, dimethyl sulfate; MNNG, N-methyl-N'-nitro- payment. This article must therefore be hereby marked "advertisement" N-nitrosoguanidine. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 5873 Downloaded by guest on September 29, 2021 5874 Biochemistry: Saparbaev and Laval Proc. Natl. Acad. Sci. USA 91 (1994) purified onto a Sephadex G-50 column (0.7 x 20 cm) equil- EDTA (buffer A), and then the cells were resuspended in 8 ibrated with 10 mM Tris HCl, pH 7.5/1 mM EDTA. The vol of buffer A and stored at -70'C. The frozen cells were polymer had a specific activity of 160,000 cpm/pg. This thawed in an ice-water bath and a lysozyme solution was substrate is hereafter referred to as [3H]dIMP M13mp8 DNA. added to a final concentration of 1 mg/ml. The suspension Alkylated DNA substrates [3H]DMS DNA and [3H]DMS was incubated at 00C for 5 min and then heated for 15 min at poly(dGC-dGC) were prepared as described using [3H]DMS 37C. The suspension was placed in an ethanol/dry-ice bath (21, 22). The specific activities of the 3H-methylated sub- and allowed to freeze for a period of 15 min. This freeze-thaw strates were 1995 cpm per pmol of methylated bases. cycle was repeated several times, after which the suspension Radioactively Labeled Double-Stranded Oligonucleotides. was centrifuged for 30 min at 30,000 rpm in a Beckman 42.1 Single-stranded oligonucleotides with or without dIMP res- rotor. The supernatant (crude lysate, 57 ml; 860 mg of idues were synthesized by E. Lescot or purchased from protein) was collected and an equal vol of 1.6% streptomycin Genset, Paris. The sequence of the oligodeoxyribonucleotide sulfate in buffer A was slowly added. After 60 min of used is 5'-AAATACATCGTCACCTGGGICATGTTGCA- incubation at 00C, the precipitate was removed by centrifu- GATCC-3'. This sequence will be referred to as Hyp2O, and gation. The clarified solution was made 1.8 M in ammonium duplex oligonucleotides will be referred to as Hyp2O/T, sulfate (46% saturation). The resulting precipitate was col- Hyp2O/G, Hyp2O/C, and Hyp2O/A when the opposite base lected by centrifugation and resuspended in 2 ml of buffer B is T, G, C, and A, respectively, in the complementary strand. (300 mM NaCl/20 mM Hepes-KOH, pH 8.0/1 mM EDTA/5 Aliquots of 30 pmol of Hyp2O oligonucleotide were 5'-end- mM 2-mercaptoethanol/0.1 mM phenylmethylsulfonyl fluo- labeled with [y32P]ATP (3000 Ci/mmol) using T4 polynucle- ride) and dialyzed against buffer B for 12 hr. The dialyzed otide kinase (New England Biolabs). Oligonucleotide du- solution (fraction II, 8 ml; 294 mg of protein) was applied to plexes were made by annealing in a mixture of a nonradio- a gel-exclusion column (2.2 x 50 cm) of AcA54 (IBF, Paris) active and complementary radioactive labeled oligonucleotide equilibrated with buffer B. Fractions containing Hyp DNA in a molar ratio of2:1 in water. The mixture was heated at 65°C glycosylase activity were pooled (fraction III, 33 ml; 60mg of for 10 min and then slowly cooled to room temperature. The protein). Fraction III (30 mg of protein) was dialyzed for 12 formation of double-stranded oligonucleotides was monitored hr against two changes of 500 ml of buffer C (as buffer B by 15% polyacrylamide gel electrophoresis under nondena- without NaCl but supplemented with 5% glycerol) and ap- turing conditions (23). plied to a Mono S FPLC HR 5/5 column (Pharmacia). The DNA Glycosylase Assays. The Hyp DNA glycosylase stan- column was rinsed with buffer C, and a gradient from 0 to 800 dard reaction mixture (200 O4) contained 2 pmol of 32p mM NaCl (15 ml) was used to develop the column (Fig. 1). 5'-end-labeled oligonucleotide duplex, 70 mM Hepes-KOH The fractions most active in Hyp DNA glycosylase were (pH 7.8), 1 mM EDTA, 5 mM 2-mercaptoethanol, 100 pg of pooled and stored at -20°C. bovine serum albumin per ml (Molecular Biology Grade, Preparation of N-Methyl-N'-nitro-N-nitrosoguanidine Boehringer Mannheim), 20 units of E. coli FPG protein (to (MNNG)-Adapted Ceils. E. coli CC106 cells were grown in LB incise DNA at abasic sites) (24), and a limiting amount of the broth to OD6w = 0.2, at which time MNNG was added to a enzyme. In the case of human, rat, and yeast 3-MeAde DNA final concentration of 0.02 mM, and cells were incubated 90 glycosylases, the incubation mixture was supplemented with min at 370C under aeration (11, 12). Cells were harvested and 100mM KCl (13, 14, 19). Incubations were for 30 min at 37°C washed two times with buffer A, resuspended in 8 vol of the unless otherwise stated. Reactions were stopped by addition same buffer, and stored at -20°C. Preparation ofcrude lysate of 12 ,ul of3 M NaCl, followed by extraction with an equal vol was made as described above. ofphenol/chloroform (1:1). The samples were centrifuged for 3 min, the aqueous phase was collected, and the DNA was RESULTS precipitated by addition of 300 ul of ethanol (-20°C). The precipitate was recovered by centrifugation, dried, and dis- Purification of E. coli Hyp DNA Glycosylase. Hyp DNA solved in 10 td offormamide, heated for 3 min at 900C, loaded glycosylase activity was purified from E. coli CC106 as onto 20%o polyacrylamide gels containing 7 M urea, and described in Materials and Methods. The Hyp DNA glyco- electrophoresed in Tris/borate/EDTA buffer. The gels were sylase activity eluted from the exclusion column as a protein subjected to autoradiography. For quantification, the bands of =30 kDa as described (5) and it coeluted with a DNA were excised and radioactivity was measured by Cerenkov glycosylase activity releasing 3-MeAde from [3H]DMS- radiation determination. treated DNA and 7-MeGua from [3H]DMS-treated poly- 3-MeAde or 7-methylguanine (MeGua) DNA glycosylase (dGC-dGC). The active fractions were further purified by assays were performed using as substrate [3H]DMS DNA or FPLC using a Mono S column. The elution profile showed 15 [3H]DMS poly(dGC-dGC) as described (13, 21). peaks of different magnitudes. As shown in Fig. 1A, Hyp HPLC Chromatography. To characterize the products of DNA glycosylase eluted as a single symmetric peak (at 0.32 the reaction, HPLC was used. Assays were performed with M NaCl) centered on peak 6 (fraction 12). 3-MeAde DNA 10,000 cpm of [3H]dIMP M13mp8 DNA incubated with 63 glycosylase eluted in two peaks (Fig. 1B). The first one, units of AlkA protein. The ethanol-soluble products dried centered on fraction 8 and eluting between peaks 1 and 2 at under vacuum were resuspended in 100 p1 of water contain- 0.22 M NaCl, is TagI [most ofthis activity, which is the major ing hypoxanthine, deoxyinosine, and dIMP (0.05 mM each) activity in E. coli (11, 12), is removed during the ammonium as authentic marker molecules. The samples were analyzed sulfate precipitation step]. The second peak of 3-MeAde by HPLC using a C(18 ,Bondapak column (Waters). The DNA glycosylase activity coelutes with Hyp DNA glycosy- column was developed isocratically at 0.8 ml/min with a lase and coincides with 7-MeGua DNA glycosylase activity, mobile phase of 20 mM (NH4)2HP04 (pH 8.5) containing 5% and, characteristic of AlkA protein, it is centered on peak 6 methanol. The radioactivity in each 0.8-ml fraction was (fraction 12). The ratios of the three activities were constant quantified by scintillation spectroscopy. across the peak. Attempts to separate these three enzymatic Enzyme Purification. Since the goal of our investigations activities using a phenyl Sepharose column, which separates was purification of hypoxanthine DNA glycosylase from E. proteins according to their hydrophobic properties, were coli, the procedure used was similar to the method of Karran unsuccessful; the three activities coeluted (data not shown). and Lindahl (5), with minor modifications (24). Briefly, 12 g In conclusion, the three different enzymatic activities had of E. coli CC106, harvested in the logarithmic growth phase, very similar, if not identical, charge and molecular mass, and was washed with 200 ml of 300 mM Tris HCI, pH 8.0/5 mM the Hyp DNA glycosylase could be the same protein as ALkA. Downloaded by guest on September 29, 2021 Biochemistry: Saparbaev and Laval Proc. Natl. Acad. Sci. USA 91 (1994) 5875
1.00 untreated wild-type E. coli CC106. However, crude extracts from cells that have been adapted by a nontoxic dose of 0 hLJ A MNNG show a detectable activity on dIMP-containing oli- Li @3 0.75 - gonucleotides. The adapted cells used showed a 5-fold in- LLI (D crC crease in 7-MeGua DNA glycosylase activity, which is char- Lii E acteristic of the AlkA protein. Z' 0.50- I CL Increase in Hyp DNA Glycosylase Activity in E. coil Strain
E Bearing the pALKiG Plasid Overproducing the AMkA Pro- a- 0.25 tein. pALK10 plasmid contains the alkA gene and allows O overproduction ofthe AlkA protein in bacteria harboring this plasmid. As shown in Fig. 2B, crude extracts from JM105 under our conditions have no detectable Hyp DNA glyco- 0.0020 5 10 15 20 25 30 35 sylase activity (as already shown for E. coli CC106 in Fig. 2A), whereas crude extracts from this strain harboring -4 pALK10 incise the oligonucleotide with good efficiency. LLI C,, m Furthermore, when the same purification procedure de- -I scribed in Fig. 1 was used to purify the Hyp DNA glycosylase uij'Li-i- 3 < ° GO from E. coli GC4802, with a mutant devoid of AlkA protein, (0>c we were unable to detect Hyp DNA glycosylase activity, 0 even after the last step of purification and concentration by w E mz the Mono S column. In a parallel experiment, the purification I m a rt-M of Hyp DNA glycosylase from the isogenic strain GC4800 I.. mT (alkA+) yielded a Hyp DNA glycosylase activity comparable LLI to that shown in Fig. 1 (data not shown). These experiments along with the previous one strongly suggest that hypoxan- 15 20 thine in duplex DNA is one of the various substrates recog- FRACTION NUMBER nized by the AlkA protein of E. coli. The Homogeneous AIkA Protein Acts on DNA Contg FIG. 1. Copurification of Hyp DNA glycosylase, 3-MeAde DNA dIMP Residues as a DNA Glycosylase. The previous results glycosylase, and 7-MeGua DNA glycosylase activity on FPLC using obtained with crude extracts show that the E. coli AlkA a Mono S HR 5/5 column. Proteins from wild-type E. coli CC106, protein is endowed with an activity recognizing dIMP resi- previously purified by gel filtration on an AcA54 column, were dues in DNA. Therefore, the ALkA protein from E. coli, chromatographed by FPLC on the cation-exchange column Mono S. purified to apparent homogeneity as judged by polyacryl- Eluted proteins were continuously monitored for absorbance at 280 amide nm and assayed for Hyp DNA glycosylase (oligonucleotide assay) gel electrophoresis (details will be published else- (i) (A) and 3-MeAde DNA glycosylase (A) and 7-MeGua DNA where), was used to characterize Hyp DNA glycosylase glycosylase (e) (B). activity. Fig. 3 shows HPLC analysis of the ethanol-soluble products obtained after incubation of M13 DNA containing Inductin of the Adaptive Response Increases the Hyp DNA dIMP residues radioactively labeled on both the sugar and the Glycosylase Activity in E. coi CC106. If Hyp DNA glycosy- base residues with AlkA protein. This result shows that there lase activity is carried out by the AlkA protein, it should also is a single peak of radioactivity eluting at the position of be induced by treatments eliciting adaptive responses (11, authentic hypoxanthine. No radioactivity is detected at the 12). As shown in Fig. 2A, under the conditions used, Hyp position of elution of deoxyinosine and dIMP. This result is DNA glycosylase activity is barely, if at all, detectable in in agreement with the results described in Fig. 2B, where we observe a band at the position of 19-mer resulting from the 1 2 3 1 2 3 4 sequential action of the ALkA protein and the apurinic/ A B apyrinidinic (AP) endonucleases present in the crude ex- tracts. 400 dIMP Hyp dino E (-S 300 1 1 FIG. 2. Hyp DNA glycosylase activity in E. coli extracts. (A) I Control or adapted cells. Crude extracts from control and adapted E. *_-/ coli CC106 cells were incubated with 32P-labeled oligonucleotides m (34-mer) containing dIMP residues at position 20. The products ofthe 200 reaction were purified and analyzed on a 7 M urea/20% polyacryl- amide gel. Lanes: 1, oligonucleotide Hyp2O/T; 2, oligonucleotide Hyp20/T incubated for 60 min in the presence of 20 lag of crude 0(X extract ofE. coli CC106 without treatment; 3, as in lane 2 but E. coli 0 cells were pretreated for 90 min with MNNG. (B) E. coli JM105 with or without plasmid pALK10 overproducing AlkA protein. Crude C o~~~~~ l extracts from E. coli JM1O5 with or without pALK10 were incubated with the duplex oligonucleotide containing dIMP residues, pro- 0 5 10 15 20 25 cessed, and analyzed as in A. Lanes: 1, oligonucleotide Hyp2O/T; 2, ELUTION TIME (min.) oligonucleotide Hyp2O/T incubated with 10 MAg of proteins from crude lysate from JM105 for 30 min; 3, as in lane 2 but using 20 Mg FIG. 3. HPLC analysis ofethanol-soluble material released by E. ofprotein; 4, as in lane 2 but using 10 Mig ofJM105 bearing pALK10. coli ALkA protein acting on M13mp8 double-stranded DNA contain- Upper arrow points to Hyp2O 34-mer; lower arrow points to cleaved ing [3H]dIMP. Standard reaction mixture was incubated with (-) or 19-mer oligodeoxyribonucleotides. For details see Materials and without (o) AlkA protein and then analyzed by HPLC. Hypoxan- Methods. thine, deoxyinosine, and dIMP elution positions are indicated. Downloaded by guest on September 29, 2021 5876 Biochemistry: Saparbaev and Laval Proc. Natl. Acad. Sci. USA 91 (1994) Incubation of the AlkA protein with Hyp20/T oligonucle- Table 1. Initial velocities of cleavage of oligonucleotide duplexes otide without treatment with FPG proteins [which nick at AP containing different bases opposite the dIMP residue by E. coli sites (24)] does not nick the oligonucleotide at the position of AlkA protein dIMP residues (data not shown). Therefore, the AlkA protein Hyp DNA glycosylase activity,* acts on dIMP residues as a DNA glycosylase. AlkA protein Substrate fmol of hypoxanthine released per min does not excise hypoxanthine from single-stranded DNA at IT 5.1 a detectable rate and does not incise the control duplex DNA, in which Hyp/T is replaced by the correct APT pair (data not I-C 4.0 shown). IG 3.8 Kinetics of Excision of Hypoxanthine from DNA Containing 1A 5.8 dIMP Residues. We have compared the kinetics ofexcision of Hyp2O 34-mer was annealed with the complementary 34-mer hypoxanthine and 3-MeAde from DNAs containing these two oligonucleotides to generate the following mismatches IT, I-A, 1
1. Table 2. Comparison of activity of human, rat, yeast, and E. coli 3-MeAde DNA glycosylases measured on substrates containing either methylated bases or deoxyinosine residues Product released by 1 unit of 3-MeAde DNA glycosylase Origin of Hyp20/T 3-MeAde DNA 3-MeAde released,* oligonucleotide glycosylase pmol/min incised,t pmol/min Human (ANPG70) 1 0.146 Human splicing variant (ANPG60) 1 0.158 Human truncated (ANPG40) 1 0.311 001 .0 Rat (ADPG) 1 0.149 1/[S] Yeast (MAG) 1 0.0004 FIG. 4. Apparent Km of E. coli Hyp DNA glycosylase for E. coli (AlkA) 1 0.0012 deoxyinosine residues in DNA. Standard enzymatic assays were *Substrate is [3H]DMS DNA and concentration of3-MeAde residues carried out by incubating AlkA protein with Hyp2O/T oligonucleo- in the incubation mixture is 10 nM. tides at the indicated concentrations ofdIMP residues ([S]) expressed tSubstrate is Hyp2O/T 34-mer and concentration ofdIMP residues is as nM. Velocity (V) is expressed as nM per min. 10 nM. Downloaded by guest on September 29, 2021 Biochemistry: Saparbaev and Laval Proc. Natl. Acad. Sci. USA 91 (1994) 5877
isolation of an activity recognizing dIMP-containing DNA; of the bases surrounding the dIMP residues in DNA has not (v) overproduction of AlkA protein parallels the overproduc- been investigated. The repair of N-methylpurines -in the tion of Hyp DNA glycosylase; (vi) homogeneous preparation dihydrofolate reductase gene in CHO cells is not strand of AlkA protein exhibits a Hyp DNA glycosylase activity. specific (30). Therefore, it is expected that repair of dIMP AlkA protein using as substrate dIMP-containing DNA residues in such an essential gene will not be strand specific. acts as a DNA glycosylase, since free hypoxanthine is Our results should lead to investigation of the biological recovered as the sole enzymatic reaction product, with no implications of deamination of adenine in vivo. detectable (3-lyase activity acting at abasic sites. The char- acteristic feature of this enzymatic reaction is that it shows a We thank C. d'Hdrin and G. Chyzak for excellent technical very high Km of 420 nM as compared with a Km of 5-7 nM assistance, Drs. T. Lindahl and G. Dianov for the gift of oligonu- when using DMS DNA as substrate. Such a large difference cleotides and for communicating a preprint, Drs. E. Seeberg and M. has also been observed for FPG protein acting on the bulky Sekiguchi for plasmids and strains, Dr. T. O'Connor for the gift of adduct imidazole ring opened form of N-hydroxy-2-amino- overproducer clones and mammalian enzyme as well as for his fluorene C8 guanine adduct: a Km of 94 nM was observed as interest in this work, Dr. S. Boiteux for bacterial strains and compared with 6 nM for imidazole ring 7-MeGua (25). The stimulating discussions, Dr. I. Felzenszwalb for initiating the prep- AlkA under aration of various substrates, Dr. E. Lescot for synthesis of oligo- observation that 1 unit of protein liberates, nucleotides, and J. Seit6 for editorial assistance. This work was similar conditions, 1 pmol of 3-MeAde from DMS DNA and supported by the Centre National de la Recherche Scientifique only 1.2 fmol of hypoxanthine under the same conditions (CNRS), the Association pour la Recherche sur le Cancer, and the when using dIMP-containing DNA (Table 2) explains why European Economic Community EV5-C91-0012 (MNLA). M.S. this activity has been difficult to purify from E. coli (5) and was the recipient of fellowships from CNRS and Ligue Nationale why enzymatic preparations of Hyp DNA glycosylase of contre le Cancer. higher specific activity were obtained from mammalian ex- tracts (2). 1. Lindahl, T. (1993) Nature (London) 362, 709-714. The association of the N-alkylpurine-DNA glycosylase 2. Karran, P. & Lindahl, T. (1980) Biochemistry 19, 6005 6011. 3. Hill-Perkins, M., Jones, M. D. & Karran, P. (1986) Mutat. Res. 162, activity with Hyp DNA glycosylase activity in the same 153-163. protein seems to be conserved during evolution, since it is 4. Schuster, H. (1960) Biochem. Biophys. Res. Commun. 2, 320-323. observed in human, rat, yeast, and E. coli enzymes. This 5. Karran, P. & Lindahl, T. (1978) J. Biol. Chem. 253, 5877-5879. result is expected since these different proteins show some 6. Myrnes, B., Guddal, P. H. & Krokan, H. (1982) Nucleic Acids Res. 10, their amino acid 3693-3701. conservation of sequences (13). 7. Dehazya, P. & Sirover, M.-A. (1986) Cancer Res. 46, 3756-3761. It appears that 3-MeAde DNA glycosylases from different 8. Dianov, G. & Lindahl, T. (1991) Nucleic Acids Res. 19, 3829-3833. origins have a broad specificity, eliminating a number of 9. Kamiya, H., Miura, H., Kato, H., Nishimura, S. & Ohtsuka, E. (1992) methylated and ethylated purine and pyrimidine bases (10- Cancer Res. 52, 1836-1839. 15, 19). Ethenoadenine is excised by ANPG protein (ref. 26; 10. Nakabeppu, Y., Miyala, T., Kondo, H., Iwanaga, S. & Sekiguchi, M. and are (1984) J. Biol. Chem. 259, 13730-13736. unpublished results); hydroxy chloroethyl purines 11. Karran, P., Hjelmgren, T. & Lindahl, T. (1982) Nature (London) 2%, excised by E. coli AlkA protein (27) and APDG rat enzyme 770-773. (unpublished results). 12. Evensen, G. & Seeberg, E. (1982) Nature (London) 296, 773-775. The implication of dIMP residues as a mutagenic lesion has 13. O'Connor, T. R. & Laval, J. (1991) Biochem. Biophys. Res. Commun. been addressed by several authors. Hill-Perkins et al. (3) have 176, 1170-1177. 14. O'Connor, T. R. & Laval, F. (1990) EMBO J. 9, 3337-3342. investigated the mutagenicity of dIMP introduced as a single 15. Berdal, K. G., Bjoras, M., Bjelland, S. & Seeberg, E. (1990) EMBO J. lesion, at a specific locus, in a M13mp9 replicative form 9, 4563-4568. molecule. They concluded that cellular Hyp DNA glycosy- 16. Boiteux, S., Huisman, 0. & Laval, J. (1984) EMBO J. 3, 2569-2573. from DNA 17. Cupples, C. G. & Miller, J. H. (1989) Proc. Natl. Acad. Sci. USA 86, lase acts inefficiently in removal of hypoxanthine 5345-5349. in vivo. The very low abundance and the very high Km of 18. Boiteux, S., O'Connor, T. R., Lederer, F., Gouyette, A. & Laval, J. AlkA protein support the fact that in E. coli in vivo, repair of (1990) J. Biol. Chem. 265, 3916-3922. deaminated adenine does not occur at a significant rate (3). 19. O'Connor, T. R. (1994) Nucleic Acids Res. 21, 5561-5569. purification of E. coli from the alkA- 20. Karran, P. (1981) in DNA Repair: A Laboratory Manual of Research Furthermore, protein Procedures, eds. Friedberg, E. C. & Hanawalt, P. C. (Marcel Dekker, strain did not allow, under our experimental conditions, New York), Vol. 1, pp. 265-273. detection of an enzymatic activity releasing hypoxanthine 21. Laval, J. (1977) Nature (London) 269, 829-833. residues from dIMP-containing DNA. The circumstances 22. Graves, R. J., Felzenszwalb, I., Laval, J. & O'Connor, T. R. (1992) J. could be different in mammalian cells, where this enzymatic Biol. Chem. 267, 14429-14435. 23. Castaing, B., Geiger, A., Seliger, H., Nehls, P., Laval, J., Zelwer, C. & activity is much higher. It could be tentatively explained by Boiteux, S. (1993) Nucleic Acids Res. 21, 2899-2905. the fact that, in higher organisms, due to the size of their 24. O'Connor, T. R. & Laval, J. (1989) Proc. Natd. Acad. Sci. USA 86, genomes, the deamination of adenine could be physiologi- 5222-5226. The excision of opposite dif- 25. Boiteux, S., Bichara, M., Fuchs, R. P. P. & Laval, J. (1989) Carcino- cally important. hypoxanthine genesis 10, 1905-1909. ferent bases by the E. coli enzyme shows no preference, 26. Singer, B., Antoccia, H., Basu, A. K., Dosanjh, M. K., Fraenkel- which is at variance with the mammalian enzymes exhibiting Conrat, H., Gallagher, P. E., Kusmierek, J. T., Qui, Z.-H. & Rydberg, a strong preference for I'T and I-G base pairs (see above B. (1992) Proc. Natl. Acad. Sci. USA 89, 9386-9390. results and ref. 8). None of these proteins acts on dIMP 27. Habraken, Y., Carter, C. A., Sekiguchi, M. & Ludlum, D. (1991) Carcinogenesis 12, 1971-1973. residues in single-stranded oligonucleotides. Studies on the 28. Martin, F. H., Castro, M. M., Aboul-ela, F. & Tinoco, I., Jr. (1985) stability ofdeoxyribonucleotide duplexes containing a deoxy- Nucleic Acids Res. 13, 8927-8938. inosine residue with each of the four normal bases show that 29. Kawase, Y., Iwai, S., Inoue, H. & Ohtsuka, E. (1986) NucleicAcids Res. the order of stability is IC > IA > IG FIT (28, 29). Thus, 19, 7727-7736. 30. Scicchitano, D. A. & Hanawalt, P. C. (1989) Proc. Natl. Acad. Sci. USA human enzyme seems to better recognize the less stable base 86, 3050-3054. pair. This is at variance with E. coli ALkA protein, which 31. Vickers, M. A., Vyas, P., Harris, P. C., Simmons, D. L. & Higgs, D. R. recognizes them almost equally well. However, the influence (1993) Proc. Natl. Acad. Sci. USA 98, 3437-3441. Downloaded by guest on September 29, 2021