Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You or Your Team.

Learn More →

Novel repair activities of AlkA (3‐methyladenine DNA glycosylase II) and endonuclease VIII for xanthine and oxanine, guanine lesions induced by nitric oxide and nitrous acid

Novel repair activities of AlkA (3‐methyladenine DNA glycosylase II) and endonuclease VIII for... ã 2002 Oxford University Press Nucleic Acids Research, 2002, Vol. 30 No. 22 4975±4984 Novel repair activities of AlkA (3-methyladenine DNA glycosylase II) and endonuclease VIII for xanthine and oxanine, guanine lesions induced by nitric oxide and nitrous acid Hiroaki Terato, Aya Masaoka, Kenjiro Asagoshi, Akiko Honsho, Yoshihiko Ohyama, 1 1 1 2 Toshinori Suzuki , Masaki Yamada , Keisuke Makino , Kazuo Yamamoto and Hiroshi Ide* Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Higashi-Hiroshima 739-8526, Japan, Institute of Advanced Energy, Kyoto University, Gokasho, Uji 611-0011, Japan and Biological Institute, Graduate School of Science, Tohoku University, Sendai 980-8578, Japan Received May 21, 2002; Revised July 18, 2002; Accepted September 24, 2002 ABSTRACT that approximately 100 C residues are deaminated per human cell per day (2). If unrepaired the resulting uracil (U) induces Nitrosation of guanine in DNA by nitrogen oxides CG®TA transitions (3). Deamination of A gives rise to such as nitric oxide (NO) and nitrous acid leads to hypoxanthine (Hx), which pairs with C and induces AT®GC formation of xanthine (Xan) and oxanine (Oxa), transitions (4). Since U and Hx are mutagenic, organisms are potentially cytotoxic and mutagenic lesions. In the equipped with repair mechanisms for these lesions. Uracil in present study, we have examined the repair capacity DNA is removed by uracil-DNA glycosylase, which is highly of DNA N-glycosylases from Escherichia coli for conserved in prokaryotic and eukaryotic organisms (5). Xan and Oxa. The nicking assay with the de®ned Hypoxanthine is generally excised from DNA by a family of methyl purine DNA glycosylases (6) and Escherichia coli has substrates containing Xan and Oxa revealed that an extra enzyme for Hx, namely endonuclease (Endo) V, AlkA [in combination with endonuclease (Endo) IV] which incises the second phosphodiester bond on the 3¢ side of and Endo VIII recognized Xan in the tested enzymes. Hx (7). The activity (V /K ) of AlkA for Xan was 5-fold max m Guanine undergoes spontaneous hydrolytic deamination to lower than that for 7-methylguanine, and that of yield xanthine (Xan), but the spontaneous deamination rate of Endo VIII was 50-fold lower than that for thymine G is lower than that of C and A (8). Therefore, so far as glycol. The activity of AlkA and Endo VIII for Xan spontaneous deamination is concerned, deamination of G is was further substantiated by the release of [ H]Xan biologically less important than that of C or A. However, from the substrate. The treatment of E.coli with nitrogen oxides such as nitric oxide (NO) and nitrous acid N-methyl-N ¢-nitro-N-nitrosoguanidine increased the (HNO ) induce deamination of DNA bases with signi®cant Xan-excising activity in the cell extract from alkA rates. In contrast to spontaneous hydrolytic deamination, G is but not alkA strains. The alkA and nei (the Endo VIII the most sensitive of the three bases to nitric oxide and nitrous gene) double mutant, but not the single mutants, acid. NO has been characterized primarily as a second messenger exerting various physiological activities (9). In exhibited increased sensitivity to nitrous acid rela- humans, 1 mmol of NO is constitutively generated per body tive to the wild type strain. AlkA and Endo VIII also per day and the amount increases by 10±100 times upon exhibited excision activity for Oxa, but the activity bacterial infection or in¯ammation. NO overproduced by was much lower than that for Xan. activated macrophages in chronically in¯amed tissues has been implicated as carcinogenic by virtue of its ability to cause DNA damage (10,11). Nitrous anhydride (N O ), formed by 2 3 INTRODUCTION autoxidation of NO, is a powerful nitrosating agent, and induces deamination of C, A and G in nucleosides and DNA, DNA that stores genetic information of cells shows certain structural instability. The DNA bases bearing an exocyclic generating U, Hx and Xan, respectively (12). Nitrous acid that amino group [adenine (A), guanine (G), cytosine (C)] undergo is formed by protonation of sodium nitrite (NaNO ), present in deamination. The rate of hydrolytic deamination is the highest food intake, also induces base deamination via N O . 2 3 for C under physiological conditions (1), and it is estimated Although nitrosation of C and A leads exclusively to U and *To whom correspondence should be addressed. Tel/Fax: +81 824 24 7457; Email: ideh@hiroshima-u.ac.jp Present address: Toshinori Suzuki, Unit of Endogenous Cancer Risk Factors, International Agency for Research on Cancer, Lyon, France Downloaded from https://academic.oup.com/nar/article-abstract/30/22/4975/2380497 by Ed 'DeepDyve' Gillespie user on 31 January 2018 4976 Nucleic Acids Research, 2002, Vol. 30 No. 22 Table 1. List of oligonucleotides used in this study Abbreviation Sequence 19T 5¢-ACAGACGCCATCAACCAGG 19TG 5¢-ACAGACGCCATgCAACCAGG COM19A 3¢-TGTCTGCGGTAGTTGGTCC PRIM15 5¢-CATCGATAGCATCCT Figure 1. Products formed by the reaction of guanine (G) with nitrogen 25G 5¢-CATCGATAGCATCCTGCCTTCTCTC oxides (NO and HNO ). 25MG 5¢-CATCGATAGCATCCTMCCTTCTCTC 25XAN 5¢-CATCGATAGCATCCTXCCTTCTCTC 25OXA 5¢-CATCGATAGCATCCTOCCTTCTCTC COM25A 3¢-GTAGCTATCGTAGGAAGGAAGAGAG Hx, respectively, that of G gives rise to not only Xan but also COM25T 3¢-GTAGCTATCGTAGGATGGAAGAGAG oxanine (Oxa) with a molar ratio of 3 (Xan):1 (Oxa) (Fig. 1) COM25G 3¢-GTAGCTATCGTAGGAGGGAAGAGAG (13). Recently, it has been shown that Xan and Oxa are formed COM25C 3¢-GTAGCTATCGTAGGACGGAAGAGAG COM30C 3¢-AAGTCGTAGCTATCGTAGGACGGAAGAGAG by nitroso group transfer to G from N-nitrosoindoles (14). Xan and Oxa are produced via the common precursor (a diazoate Tg, thymine glycol; M, 7-methylguanine; X, xanthine; O, oxanine. Base derivative of the 2-amino group of G), but formation of Oxa lesions and paired bases are underlined. involves deamination of the 2-amino group followed by rearrangement of the ring atoms (15). The biochemical and genotoxic effects of Xan and Oxa have been assessed nei alkA phenotypes were con®rmed by measuring Endo VIII previously by several studies. Substitution of Xan or Oxa for and Endo VIII/AlkA activities and the strains were designated G in duplex DNA results in large decreases in the helix KY100 (MV1161 + nei) and KY101 (MV1571 + nei), stability (16). Xan in template DNA directs incorporation of T respectively. Endo III, Endo VIII, Endo IV, formamido- as well as C during DNA replication (17,18). Similarly, pyrimidine DNA glycosylase (Fpg/MutM) and AlkA were 2¢-deoxyribonucleoside 5¢-triphosphates of Xan (dXTP) and puri®ed from E.coli strains that overproduced these enzymes Oxa (dOTP) are incorporated opposite template T as well as C (24,25). Escherichia coli uracil DNA glycosylase (Ung) and by DNA polymerase, though their incorporation ef®ciencies exonuclease (Exo) III were purchased from New England are much lower than dGTP (19). In addition, Xan but not Oxa Biolabs. Escherichia coli DNA polymerase I Klenow frag- can be spontaneously converted to a cytotoxic and mutagenic ment (Pol I Kf) and T4 polynucleotide kinase were obtained AP (apurinic/apyrimidinic) site due to its labile N-glycosidic from Life Technologies. 2¢-Deoxycytidine 5¢-triphosphate bond (16). Thus, Xan may show additional biological effects (dCTP), thymidine 5¢-triphosphate (dTTP) and [g- P]adeno- after conversion to an AP site. Recently, Oxa has been sine 5¢-triphosphate (ATP) (110 TBq/mmol) were from shown to form a covalently bound adduct with glycine, Amersham Biosciences. 2¢-[8- H]Deoxyguanosine 5¢-triphos- suggesting a novel cytotoxic/genotoxic mechanism for this phate (dGTP) (37 MBq/244 pmol) was purchased from NEN lesion (20). These data combined together indicate the Life Science Products. Guanine and Xan were purchased from necessity of repair capacities for Xan and Oxa in cells. Sigma. However, studies on the enzymatic repair of these lesions are extremely limited (21). Oligonucleotides In the present work, we have examined the repair capacity Oligonucleotides without modi®ed bases were purchased from of E.coli base excision repair enzymes for Xan and Oxa using Espec Oligo Service and puri®ed by reversed phase HPLC. de®ned oligonucleotide substrates containing these lesions. Preparation of 25MG and 19TG which contain a single We report here that, among the enzymes tested, AlkA and 7-methylguanine (7mG) and thymine glycol (Tg), respect- Endo VIII exhibit repair activities for Xan and Oxa in a paired ively, were reported previously (26,27). The sequences of the base-dependent manner and the repair ef®ciency for Oxa is oligonucleotides used in this study are shown in Table 1. much lower than for Xan. Induction of the alkA gene encoding AlkA protein in E.coli cells results in an increased releasing Preparation of dXTP and dOTP and their incorporation activity of Xan. Furthermore, E.coli de®cient in both AlkA into oligonucleotides and Endo VIII, but not either of them alone, exhibits increased sensitivity to nitrous acid. 2¢-Deoxyxanthosine 5¢-triphosphate (dXTP) and 2¢-deoxy- oxanosine 5¢-triphosphate (dOTP) were synthesized from dGTP. dGTP (1 mM) was incubated with 100 mM NaNO at 37°C in 3 M acetate buffer (pH 3.7) for 2 h, and the resulting MATERIALS AND METHODS dXTP and dOTP were puri®ed by reversed phase HPLC. The Strains, enzymes and chemicals detailed procedures and characterization of dXTP and dOTP Escherichia coli MV1161 (thr1, ara14, leuB6, D(gpt-proA)62, were reported previously (19). [ H]dXTP was prepared 3 3 lacY1, tsx33, supE44, galK2, hisG4, rfbD1, mgl51, rpsL31, from [ H]dGTP in an essentially similar manner. [ H]dGTP kdgK51, xyl5, mtl1, argE3, thi1, rfa550) and MV1571 (37 MBq/244 nmol, diluted with cold dGTP) was incubated [alkA51::Mud1 (Amp lac) in MV1161] were laboratory stocks with 100 mM NaNO in 1 M acetate buffer (pH 3.7, 100 ml) at (22). To construct Endo VIII mutants, MV1161 and MV1571 37°C for 2 h. [ H]dXTP was puri®ed by reversed phase HPLC. were infected with P1 phage carrying the Dnei::Km allele Oligonucleotide substrates containing Xan and Oxa were derived from NKJ1003 (23) and were selected for the prepared by DNA polymerase reactions. PRIM15 was 5¢-end kanamycin (Km)-resistant phenotype. The resultant nei and labeled using [g- P]ATP and T4 polynucleotide kinase and Downloaded from https://academic.oup.com/nar/article-abstract/30/22/4975/2380497 by Ed 'DeepDyve' Gillespie user on 31 January 2018 Nucleic Acids Research, 2002, Vol. 30 No. 22 4977 puri®ed by a Sep-pak cartridge (Waters) (28). PRIM15 (100 fmol) were incubated with AlkA (600 fmol) and Ung (50 pmol) and COM25C (100 pmol, 2-fold molar excess) in (170 mU) in buffer D (10 ml) and buffer E (10 ml), respectively, 10 mM Tris±HCl (pH 7.5) and 25 mM NaCl were heated at at 37°C for 30 min. The composition of buffer D was 50 mM 90°C for 10 min and then annealed at room temperature. HEPES-KOH (pH 7.5), 1 mM EDTA and 5 mM 2-mercapto- PRIM15/COM25C (25 pmol) in the polymerase buffer ethanol and that of buffer E was 20 mM Tris±HCl (pH 8.0), (200 ml) were incubated with dXTP or dOTP (both 20 nmol) 1 mM EDTA and 1 mM dithiothreitol. After incubation, the and Pol I Kf (25 U) at 25°C for 5 min to introduce a single substrates were puri®ed by phenol extraction and ethanol residue of Xan or Oxa opposite C (16th position from the 3¢ precipitation. The puri®ed substrates were incubated further terminus in COM25C). The polymerase buffer consisted of with Endo IV (120 fmol) at 37°C for 30 min to incise abasic 66 mM Tris±HCl (pH 7.0), 1.5 mM 2-mercaptoethanol and sites. The sample was mixed with gel loading buffer and 6.6 mM MgCl . Then dCTP and dTTP (10 nmol each) were separated by 16% denaturing PAGE. The radioactivity of added to the reaction mixture to extend the primer fully. The products was analyzed on a BAS2000 phosphorimaging oligonucleotides containing Xan and Oxa were designated analyzer (Fuji Film). Paired base effects on the activity of 25XAN and 25OXA, respectively. The duplexes (25XAN/ AlkA and Endo VIII for Xan and Oxa were measured using COM25C and 25OXA/COM25C) were puri®ed by phenol 25XAN/COM25N and 25OXA/COM25N (N = A, G, C or T) extraction, ethanol precipitation and gel ®ltration on a as substrates. The reactions were performed in a manner Sephadex G-25 column (1 ml). The site-speci®c introduction essentially similar to those described above. The amounts of of Xan and Oxa into 25XAN and 25OXA, respectively, was the substrates (25XAN and 25OXA) and the enzymes (AlkA con®rmed by the heat/acid and ammonia/piperidine treat- and Endo VIII) were 100 fmol and 300 fmol±3 pmol, respectively. ments (see Results). For preparation of oligonucleotide substrates containing Xan:N and Oxa:N pairs (N = A, G, C, For analysis of the kinetic parameters of AlkA, 25XAN/ T), the DNA polymerase reaction was performed in a similar COM25C and 25MG/COM25C (50±1000 fmol) were incu- manner except that COM30C was used as a template in place bated with 300 fmol of AlkA at 37°C for 10 min in buffer D of COM25C. This facilitated the subsequent electrophoretic (10 ml), and then the products were treated with Endo IV as separation of 25XAN and 25OXA from the template described above. For Endo VIII, 25XAN/COM25C and 19TG/ (COM30C). After the reaction, 25XAN and 25OXA were COM19A (50±1000 fmol) were incubated with Endo VIII puri®ed by 16% denaturing polyacrylamide gel electro- (300 fmol) at 37°C for 15 min (25XAN/COM25C) and 2 min phoresis (PAGE), extracted from the gel and desalted by a (19TG) in buffer A (10 ml). Reaction products were separated Sep-pak cartridge. Puri®ed 25XAN and 25OXA were and quanti®ed as described above. The parameters V and max annealed with COM25N (N = A, G, C, T). 25XAN containing K were evaluated from Michaelis±Menten plots using a [ H]Xan was prepared as described above using PRIM15/ hyperbolic curve ®tting program. COM25C, [ H]dXTP and Pol I Kf for the initial polymeriza- Release assays of Xan with AlkA and Endo VIII 3 3 tion reaction. 25G containing [ H]G in place of [ H]Xan was also prepared using [ H]dGTP. To measure the N-glycosylase activity of AlkA, 25XAN/ COM25C and 25G/COM25C (both 2.25 pmol) containing Chemical cleavage reactions of oligonucleotides 3 3 [ H]Xan and [ H]G, respectively, were incubated with AlkA 25XAN, 25OXA and 25G (all P-labeled at the 5¢ end, (3 pmol) at 37°C for 30 min in buffer D (20 ml). The reaction 100 fmol) were heated at 90°C for 30 min under acidic with Endo VIII (6 pmol) was performed in a similar manner conditions (pH 3.5, adjusted by acetic acid) and the solution using buffer A. After the reaction, the sample was loaded onto was evaporated to dryness. Alternatively, the oligonucleotides a Sephadex G-25 ®ne gel ®ltration column (f 3 3 300 mm) to were treated ®rst by 30% ammonia at 65°C for 4 h. After separate the released product and the oligonucleotides. The removing ammonia by evaporation, the samples were treated column was eluted by water and the eluent was collected every by 10% piperidine at 90°C for 30 min and evaporated to 100 ml. Aliquots of the collected fractions were subjected to dryness. The products from both treatments were mixed with liquid scintillation counting. The oligonucleotides and the gel loading buffer [95% (v/v) formamide, 20 mM EDTA, released product were eluted in fractions 12±15 and 20±25, 0.1% (w/v) xylene cyanol and 0.1% (w/v) bromophenol blue] respectively, under these conditions. The pooled fractions and analyzed by 16% denaturing PAGE. containing the released product were evaporated to dryness and resuspended in a small volume of water. The sample was Nicking assays for the activity to Xan and Oxa analyzed by reversed phase HPLC. The HPLC system In the reaction with Fpg, Endo III and Endo VIII (all consisted of Hitachi L-6200 pumps equipped with a reversed N-glycosylase/AP lyase), 25G/COM25C, 25XAN/COM25C phase WS-DNA column (f 4.6 3 150 mm, Wako) and a and 25OXA/COM25C (100 fmol) were incubated with the Hitachi L-4200H UV-VIS detector. Elution was carried out enzyme (600 fmol) in buffer A (10 ml) at 37°C for 30 min. with a linear gradient of acetonitrile (0±20%, v/v) in 20 mM Buffer A comprised 10 mM Tris±HCl (pH 7.5), 1 mM EDTA sodium phosphate buffer (pH 5.0) at a ¯ow rate 0.8 ml/min. and 100 mM NaCl. The reactions with Endo IV (120 fmol) and The column eluent was collected every 20 s and was subjected Exo III (0.1 U) (both AP endonucleases) were similarly to liquid scintillation counting. carried out in buffer B (10 ml) and C (10 ml), respectively. The Release assays of Xan with crude cell extracts composition of buffer B was 10 mM Tris±HCl (pH 7.5), 1 mM EDTA and 50 mM NaCl and that of buffer C was 10 mM Escherichia coli MV1161 (wild type) and MV1571 (alkA) Tris±HCl (pH 7.5), 2 mM CaCl and 1 mM EDTA. In the were cultivated in LB media (250 ml) in the absence reaction with simple DNA glycosylases, the substrates (MV1161) and presence (MV1571) of 50 mg/ml ampicillin Downloaded from https://academic.oup.com/nar/article-abstract/30/22/4975/2380497 by Ed 'DeepDyve' Gillespie user on 31 January 2018 4978 Nucleic Acids Research, 2002, Vol. 30 No. 22 at 37°C for 3 h under aeration. Depending on the experiments, N-methyl-N ¢-nitro-N-nitrosoguanidine (MNNG) (®nal con- centration 20 mM) was added into the culture media and incubation was continued for further 1.5 h. After recovering cells by centrifugation, the cell pellet was suspended in 10 ml of 300 mM Tris±HCl (pH 8.0), 5 mM EDTA and 1 mg/ml lysozyme, and was lyzed by repeated freeze/thaw cycles. The lyzed cells were centrifuged at 43 000 g for 1 h at 4°C and the supernatant was recovered. The proteins in the supernatant were collected by ammonium sulfate precipitation (60% saturation) and were resuspended in 10 mM Tris±HCl (pH 7.5) and 1 mM EDTA (TE buffer). After dialysis against TE buffer at 4°C, the cell extract was used for the release assay of [ H]Xan. The protein concentration was determined by a BCA protein assay reagent (Pierce) using BSA as a standard. The release assay was performed as described for AlkA using 5 mg of protein. Figure 2. Chemical cleavage of oligonucleotides containing G (25G), Xan Nitrous acid sensitivity of alkA and nei E.coli strains (25XAN) and Oxa (25OXA). 5¢- P-labeled 25G, 25XAN and 25OXA were A modi®ed procedure by Miller (29) was used. Escherichia heated at 90°C for 30 min in an acidic aqueous solution (pH 3.5, adjusted coli strains, MV1161 (wild type), MV1571 (alkA), KY100 by acetic acid) (lanes 3, 6 and 9). Alternatively they were incubated ®rst in 30% (v/v) ammonia at 65°C for 4 h, and then in 10% (v/v) piperidine at (nei) and KY101 (alkA nei) were cultured overnight in 1 ml of 90°C for 30 min as described in Materials and Methods (lanes 4, 7 and 10). LB media with 50 mg/ml of appropriate antibiotics (ampicillin The samples were separated by 16% denaturing PAGE. Lane 1 shows a 5¢- for MV1571, kanamycin for KY100, and ampicillin and P-labeled marker (PRIM15). The bands of b-elimination, d-elimination kanamycin for KY101) at 37°C. These cultures were diluted and 3¢-OH products are indicated by arrows. into 100 ml of LB media with or without antibiotics and cells were grown to a logarithmic phase (OD = 0.6) at 37°C. After washing in acetate buffer (0.1 M, pH 4.6), cells were described in Materials and Methods. PAGE analysis of the incubated in NaNO (0±30 mM) in 5 ml of acetate buffer treated oligonucleotides showed that 25XAN was speci®cally (0.1 M, pH 4.6) at 37°C for 10 min. To stop incubation, an cleaved by the heat/acid treatment (Fig. 2, lane 9) but not the equal volume of minimal A medium [60.3 mM K HPO , 2 4 ammonia/piperidine treatment (Fig. 2, lane 10). According to 33.1 mM KH PO , 7.6 mM (NH ) SO and 1.7 mM trisodium 2 4 4 2 4 the gel mobility of the products, the cleaved products were citrate] was added. After washing in minimal A medium 15mers with different 3¢ terminal modi®cations resulting twice, the cell suspension was appropriately diluted and from breakdown of an AP site (a mixture of b-elimination, incubated overnight on LB plates at 37°C. The colonies were d-elimination and 3¢-OH products). Conversely, 25OXA was scored for surviving fractions. It is noted that the viability of stable in the heat/acid treatment (Fig. 2, lane 6) but was all the test strains decreased to ~10% after incubation with speci®cally cleaved by the ammonia/piperidine treatment acetate buffer (0.1 M, pH 4.6) alone. Thus, variations in (Fig. 2, lane 7). The products were 15mers bearing 3¢- viability depending on the strain were virtually negligible phosphate (d-elimination product) and 3¢-OH groups. 25G was in the incubation with acetate buffer alone. virtually intact in both treatments (Fig. 2, lanes 3 and 4). These results indicate that Xan and Oxa were site-speci®cally introduced into 25XAN and 25OXA, respectively, by the RESULTS DNA polymerase reaction. Site-speci®c introduction of Xan and Oxa into Repair activities of AlkA and Endo VIII for Xan and oligonucleotides Oxa Xan and Oxa were site-speci®cally incorporated into the The repair activities of various E.coli DNA repair enzymes for oligonucleotide substrates (25XAN and 25OXA, Table 1) by Xan and Oxa were examined using the nicking assay with the DNA polymerase reaction using dXTP and dOTP as 25XAN/COM25C and 25OXA/COM25C as substrates. The substrates. The N-glycosidic bond of Xan is fairly labile so enzymes tested included three bifunctional glycosylases that heat/acid treatment causes depurination, generating an AP containing N-glycosylase/AP lyase activities (Fpg, Endo III site. On the other hand, the N-glycosidic bond of Oxa is as and Endo VIII), two monofunctional N-glycosylases (AlkA stable as that of G to the heat/acid treatment. However, Oxa and Ung) and two AP endonucleases (Endo IV and Exo III). contains an O-acylisourea structure in the six-membered ring Among the enzymes tested, AlkA and Endo VIII recognized (1,3-oxazine ring) that undergoes a ring-opening reaction at Xan. The results of PAGE analysis of the reaction products of alkaline pH (pK = 9.4) (16). An extensive treatment of the AlkA and Endo VIII are shown in Figure 3. The treatment of ring-opened structure of Oxa induces decomposition of the 25XAN by AlkA (followed by Endo IV) resulted in a clear Oxa ring, though the ®nal decomposition product has not been band of a 3¢-OH product arising from cleavage at the identi®ed yet. Knowing the chemistry of Xan and Oxa, 25XAN and 25OXA prepared by the DNA polymerase introduced Xan site (Fig. 3A, lane 7). Similarly, the treatment reaction were treated by heat/acid or ammonia/piperidine as with Endo VIII resulted in a d-elimination product (Fig. 3B, Downloaded from https://academic.oup.com/nar/article-abstract/30/22/4975/2380497 by Ed 'DeepDyve' Gillespie user on 31 January 2018 Nucleic Acids Research, 2002, Vol. 30 No. 22 4979 Since Xan appeared to be a fair substrate of AlkA and Endo VIII, their kinetic parameters for Xan were determined from Michaelis±Menten plots and the results were compared with those for 7mG (AlkA) and Tg (Endo VIII). The K values of AlkA for 7mG and Xan were 31 and 53 nM, respectively (Table 2). The V values were 2.1 and 0.72 nM/min for 7mG max and Xan, respectively. These results indicated that AlkA had almost comparable af®nities (K ) for 7mG and Xan, but the catalytic rate for Xan was somewhat lower than for 7mG. The difference in the reaction ef®ciencies (V /K ) between 7mG max m and Xan was only 5-fold. Considering that 7mG is a physiological substrate of AlkA (30) and the relatively small difference in the reaction ef®ciencies, Xan is a fairly good substrate for AlkA. In contrast, Endo VIII showed a signi®- cantly lower reaction ef®ciency for Xan than for Tg, a physiological substrate of Endo VIII (31±33). The difference in the V /K values was ~50-fold. In terms of af®nity (K ), max m m Xan and Tg differed by 2.6-fold (124 versus 47 nM) and of catalytic rate (V ) by 19-fold (0.94 versus 18 nM/min). max Thus, Xan is a relatively poor substrate among those recognized by Endo VIII. N-glycosylase activity of AlkA and Endo VIII for Xan To con®rm the activity of AlkA and Endo VIII for Xan further, the N-glycosylase activity of both enzymes was examined by the release assay using 25XAN/COM25C containing [ H]Xan. After enzymatic treatment, the released product was separated from the substrate by gel ®ltration. Analysis of the radio- activity of the fraction from the gel ®ltration column showed that there was signi®cant release of the tritium count when Figure 3. Reaction products formed in the treatment of substrates contain- 25XAN/COM25C was incubated with AlkA and Endo VIII. ing Xan (25XAN) and Oxa (25OXA) by AlkA and Endo VIII. (A) AlkA However, the released tritium count was negligible when reactions. 100 fmol of 25G/COM25C, 25XAN/COM25C and 25OXA/ 3 3 control 25G/COM25C containing [ H]G instead of [ H]Xan COM25C (25G, 25XAN and 25OXA were 5¢- P labeled) were incubated was incubated with AlkA and Endo VIII. This was also the with 600 fmol of AlkA at 37°C for 30 min. After incubation, the sample was extracted with phenol and DNA was recovered by ethanol precipitation. case when 25XAN/COM25C was incubated with the reaction The sample was treated further with 120 fmol of Endo IV at 37°C for buffer alone. The pooled gel ®ltration fractions containing the 30 min. (B) Endo VIII reactions. 100 fmol of 25G/COM25C, 25XAN/ released product were further analyzed by reversed phase COM25C and 25OXA/COM25C were incubated with 600 fmol of Endo HPLC. In the HPLC analysis of the product released by AlkA VIII at 37°C for 30 min. In panels (A) and (B), the sample was separated by 16% denaturing PAGE. The substrates and enzymes used are indicated and Endo VIII, the major peak of the tritium count was eluted on the top of the gels. Lane 1 shows a 5¢- P-labeled marker (PRIM15). at 10.4 min (Fig. 4B and C) where authentic Xan (unlabeled) was eluted (Fig. 4A). Authentic G was eluted at 8.7 min under these conditions. Accordingly, AlkA and Endo VIII acted as lane 7). The observed incision products were consistent with N-glycosylases for 25XAN/COM25C, releasing a free base of the known catalytic modes of AlkA (+ Endo IV) and Endo VIII. Xan from the substrate. Although incision products of 25OXA by AlkA (+ Endo IV) Repair capacity of E.coli cell extracts for Xan and Endo VIII were barely detected with the same amount (600 fmol) of the enzymes as used for 25XAN (Fig. 3A and B, The repair capacity of the E.coli cell extracts for Xan was lane 5), treatment with an excessive amount of AlkA and Endo measured by the release assay. 25XAN/COM25C containing VIII (both 3 pmol) revealed their activity for Oxa (see Paired [ H]Xan was incubated with the cell extracts from E.coli base effects on the repair activity for Xan and Oxa). MV1161 (wild type) and MV1571 (alkA), and the released Table 2. Kinetic parameters of AlkA and Endo VIII for Xan b ±1 Enzyme Substrate K (nM) V (nM/min) V /K (min ) m max max m ±2 AlkA Xanthine 53 6 24 0.72 6 0.04 1.4 3 10 (1) ±2 7-Methylguanine 31 6 2 2.1 6 0.4 6.8 3 10 (4.9) ±3 Endo VIII Xanthine 124 6 33 0.96 6 0.26 7.6 3 10 (1) ±1 Thymine glycol 47 6 10 18 6 5 3.8 3 10 (50) The values are derived from three independent experiments and standard deviations are also indicated. The values in parentheses indicate relative values of V /K . max m Downloaded from https://academic.oup.com/nar/article-abstract/30/22/4975/2380497 by Ed 'DeepDyve' Gillespie user on 31 January 2018 4980 Nucleic Acids Research, 2002, Vol. 30 No. 22 Figure 5. Xan-releasing activity of the extracts from wild type and alkA mutant E.coli cells. The cell extracts were prepared from E.coli MV1161 (wild type) and MV1571 (alkA) treated without and with MNNG, respec- tively, as described in Materials and Methods. 25XAN/COM25C containing [ H]Xan (2.25 pmol) was incubated with 5 mg of the cell extracts at 37°C for 30 min. After incubation, the sample was loaded on to a Sephadex G-25 column and eluted with water. The amount of [ H]Xan released was counted on a liquid scintillation counter. Open columns, cells without MNNG treat- ment; closed columns, cells with MNNG treatment. The data are averages of three independent experiments. Standard deviations are indicated with error bars. Figure 4. N-glycosylase activity assays of AlkA and Endo VIII for Xan. (A) HPLC separation of authentic guanine (G) and Xan. Analysis was per- (Fig. 6A, lane 3). The activities for both pairs were compar- formed as described in Materials and Methods. (B) HPLC analysis of [ H]Xan released by AlkA. 2.25 pmol of 25XAN/COM25C containing able. The Xan:G and Xan:T pairs were very poor substrates of [ H]Xan was incubated with 3 pmol of AlkA at 37°C for 30 min. The AlkA (Fig. 6A, lanes 7 and 9). The order of activity for Xan released H-labeled material was separated from DNA by a Sephadex G-25 was C = A >> G > T with respect to the paired base. Unlike column. The column fractions containing the released H-labeled material AlkA, Endo VIII recognized Xan:C and Xan:G pairs with were pooled and evaporated. The sample was resuspended in a small volume of water and was subjected to HPLC analysis. HPLC analysis was comparable ef®ciency (Fig. 6B, lanes 7 and 10). The Xan:A performed as described in panel (A). (C) HPLC analysis of [ H]Xan and Xan:T pairs were very poor substrates. The order of released by Endo VIII. The experiment was performed in a similar manner activity was C = G >> A > T with respect to the paired base. using 6 pmol of Endo VIII. Compared to the reaction with Xan, a large excess of the enzymes (5- or 10-fold) and a long incubation time (1 h) were required to detect the activity for Oxa. For Oxa, AlkA and tritium count was measured after separation on a gel ®ltration Endo VIII exhibited a similar preference of the paired bases. column. However, no signi®cant difference in released The pairs of Oxa:A (Fig. 7, lanes 4 and 5), Oxa:C (Fig. 7, lanes radioactivity was observed between wild type and alkA 8 and 9) and Oxa:G (lanes 12 and 13) were comparable mutant cells (Fig. 5). It is possible that the basal level of substrates for AlkA and Endo VIII, whereas an Oxa:T pair was AlkA protein in the wild type cell was so low that the activity recognized poorly by the enzymes (Fig. 7, lanes 16 and 17). difference between the wild type and alkA mutant cells could Therefore, the order of the activity of both enzymes was be within an experimental ¯uctuation. The alkA gene is C = G = A > T with respect to the paired base. inducible by alkylating agents (34). Therefore, MV1161 and MV1571 cells were treated with MNNG, and the cell extracts Nitrous acid sensitivities of E.coli strains de®cient in from the two strains were subjected to the release assay. After AlkA and Endo VIII MNNG treatment, the wild type cell extract showed a 4-fold increase in Xan-releasing activity in comparison with the cell To assess the role of AlkA and Endo VIII in the repair of Xan extract without the MNNG treatment (Fig. 5). In contrast, such and Oxa in E.coli cells, the sensitivity of E.coli strains induction was not observed with the alkA mutant treated with MV1161 (wild type), MV1571 (alkA), KY100 (nei) and MNNG (Fig. 5). The results obtained with MNNG-treated KY101 (alkA nei) to nitrous acid was determined by treating cells were consistent with the data obtained with puri®ed them with sodium nitrite (NaNO ) pH 4.6 (29) under selective AlkA. conditions (i.e. in the presence of appropriate antibiotics). The single mutants defective in either the alkA or nei gene showed Paired base effects on the repair activity for Xan and nitrous acid sensitivities similar to those of the wild type cell. Oxa On the other hand, the alkA nei double mutant exhibited increased sensitivity (Fig. 8), implying a degenerative role of Xan and Oxa are potentially mutagenic lesions and pair with C AlkA and Endo VIII in counteracting the cytotoxic effect of and T during DNA replication (16±18). Thus, the ability of nitrous acid. In the above NaNO treatment, E.coli strains AlkA and Endo VIII to recognize Xan and Oxa paired with MV1161, MV1571 and KY100 were grown in the absence or different bases was examined. The activity was measured by the nicking assay using 25XAN/COM25N and 25OXA/ presence of one antibiotic (ampicillin or kanamycin), while COM25N (N = A, G, C, T) as substrates. AlkA recognized strain KY101 was grown in the presence of two antibiotics not only a Xan:C pair (Fig. 6A, lane 5) but also a Xan:A pair (ampicillin and kanamycin). The differences in growth Downloaded from https://academic.oup.com/nar/article-abstract/30/22/4975/2380497 by Ed 'DeepDyve' Gillespie user on 31 January 2018 Nucleic Acids Research, 2002, Vol. 30 No. 22 4981 Figure 7. Activities of AlkA and Endo VIII for Oxa paired with different bases. 25OXA/COM25N (N = A, G, C, T) (100 fmol) was incubated with 3 pmol of AlkA or Endo VIII for 1 h and products were analyzed as described in Figure 6. The substrates (base pairs) and enzymes used were indicated on the top of the gels. Lane 1 shows a 5¢- P-labeled marker (PRIM15). Figure 6. Activities of AlkA and Endo VIII for Xan paired with different Figure 8. Sensitivity of E.coli strains to nitrous acid. Escherichia coli cells bases. (A) AlkA reactions. 25XAN/COM25N (N = A, G, C, T) (100 fmol) pro®cient and de®cient in AlkA (encoded by the alkA gene) or Endo VIII was incubated with 300 fmol of AlkA at 37°C for 20 min followed by (encoded by the nei gene) were treated with the indicated concentrations of 120 fmol of Endo IV at 37°C for 30 min. (B) Endo VIII reactions. 25XAN/ NaNO in acetate buffer (pH 4.6) and the survival fractions were deter- COM25N (N = A, G, C, T) (100 fmol) was incubated with 600 fmol of 2 mined as described in Materials and Methods. Open circles, MV1161 (wild Endo VIII at 37°C for 1 h or 120 fmol of Endo IV at 37°C for 30 min. In type); closed circles, MV1571 (alkA); open triangles, KY100 (nei); closed (A) and (B), products were analyzed by 16% denaturing PAGE. The triangles, KY101 (alkA nei). The data are averages of three independent substrates (base pairs) and enzymes used are indicated on the top of the experiments. Standard deviations are indicated with error bars. gels. Lane 1 shows a 5¢- P-labeled marker (PRIM15). The 3¢-OH and d-elimination products are indicated by arrows. conditions (i.e. selective pressure) might have created an oxides such as NO and nitrous acid. For this purpose, we advantage for the strains MV1161, MV1571 and KY100 over prepared de®ned oligonucleotide substrates containing Xan the strain KY101 with respect to survival. Thus, NaNO and Oxa by DNA polymerase reactions with dXTP and dOTP. treatment was also performed in the absence of antibiotics by It has been reported that dXTP is a poor substrate of DNA taking advantage of the fact that the chromosomal markers for polymerase I (35). Our previous study has also shown that antibiotic resistance (ampicillin and kanamycin in this study) dOTP as well as dXTP are poorly utilized by DNA polymerase I are rarely lost even in the absence of selection pressure. relative to dGTP (19). However, the low but signi®cant However, the results obtained without antibiotics were usability of dXTP and dOTP by DNA polymerase I was good essentially similar to those obtained with antibiotics, hence enough for targeted incorporation of the nucleotides under ruling out the effects of selection pressure on survival appropriate DNA polymerase reaction conditions (Fig. 2). measurements. Among the seven enzymes tested, AlkA and Endo VIII recognized Xan and Oxa, though their activity for Oxa was lower than for Xan. Analysis of enzymatic parameters DISCUSSION revealed that the activity (V /K ) of AlkA for Xan is one- max m In the present study, we have screened the repair activity of ®fth of that for 7mG, a physiological substrate of AlkA E.coli enzymes for Xan and Oxa formed from G by nitrogen (Table 2). AlkA recognizes a variety of alkylated purine and Downloaded from https://academic.oup.com/nar/article-abstract/30/22/4975/2380497 by Ed 'DeepDyve' Gillespie user on 31 January 2018 4982 Nucleic Acids Research, 2002, Vol. 30 No. 22 pyrimidine lesions (34). It has been shown that 5-formyluracil complexed covalently to DNA have recently been solved and fragmented thymine residues are also substrates of AlkA (44,45). Although the overall structures of Endo VIII and Fpg (24,36±38). Thus, AlkA accepts structurally diverse base resemble each other, there are three dissimilar patches where lesions as substrates. Common features of the substrates amino acids are not conserved between the two enzymes. One recognized by AlkA are weak N-glycosidic bonds and explicit of them coincides with putative binding pockets for the or implicit positive charges induced in the base (24). A everted base. Thus, interactions in the dissimilar binding plausible catalytic mechanism of AlkA has been proposed pocket are likely to account for the distinct damage speci®city based on these features and the three-dimensional structure of of Endo VIII and Fpg. AlkA (39±41). In view of the weak N-glycosidic bond of Xan The activities of AlkA and Endo VIII for Xan and Oxa and the capacity of the active site of AlkA, it seems reasonable showed dependence on the base opposite the lesions, though that AlkA recognizes Xan as a substrate. Oxa was also a the activity for Oxa was consistently much lower than for Xan. substrate of AlkA but recognized poorly. The relative For Xan, the activity decreased in the following order of the hydrolysis rate of the N-glycosidic bond of Oxa is 44-fold paired base: C = A >> G > T for AlkA and C = G >> A > T for lower than that of Xan (16). Therefore, it is very likely that Endo VIII (Fig. 6). For Oxa, the order was C = G = A > T for such a difference affected the hydrolysis of the N-glycosidic both AlkA and Endo VIII (Fig. 7). Considering the base bond by AlkA and slowed down the reaction of Oxa relative pairing capacity of Xan and Oxa, C and T are the most to Xan. biologically relevant paired bases. The Xan:C and Oxa:C pairs The cell extracts from the E.coli strains pro®cient and can be formed by the reaction of a G:C pair with NO and de®cient in the alkA gene showed no signi®cant difference in nitrous acid, and Xan:T and Oxa:T pairs by misincorporation Xan-releasing activity. Possibly, the basal level of AlkA of T opposite these lesions during DNA replication (16±18). protein present in wild type cells was too low to exhibit Excision of Xan and Oxa from Xan:C and Oxa:C pairs, detectable activity over the experimental ¯uctuation in the respectively, by AlkA and Endo VIII and subsequent repair present assay. Although the attempt to show the difference in synthesis leads to restoration of genetic information, thus error Xan-releasing activity between wild type and alkA mutant free. Conversely, excision of Xan and Oxa from Xan:T and cells was unsuccessful, the repair activity for Xan in the wild Oxa:T pairs, respectively, results in mutation ®xation and type cell was clearly increased when the cell was treated with induces G:C®A:T transitions if it occurs in cells. According MNNG (Fig. 5). Such an increase was not observed with the to the present results (Figs 6 and 7), AlkA and Endo VIII alkA mutant when it was treated in the same manner, recognized Xan:C and Oxa:C pairs relatively well but Xan:T indicating that the induction of the repair activity for Xan in and Oxa:T pairs were poor substrates for the enzymes. Thus, cells was dependent on the alkA gene. These results suggest the poor activity of AlkA and Endo VIII for Xan:T and Oxa:T that AlkA can at least participate in the repair of Xan when it is pairs seems to be consistent with avoidance of mutation present at the induced level. The alkA gene is under the control ®xation in cells. This is in contrast to the preference of E.coli of the ada gene and inducible by the alkylating agent such as Endo V, which recognizes all four Xan:N pairs (N = A, G, C, MNNG (34). However, no information is currently available T) with a preference for the Xan:T pair (21). Endo V was on whether NO or nitrous acid induces the alkA gene through originally found as a deoxyinosine endonuclease that incises this pathway. the second phosphodiester bond on the 3¢ side of deoxyinosine In this study, it has also been shown that Endo VIII has (a deoxyribonucleoside form of Hx) (7). More recently it has repair activity for Xan. The activity of Endo VIII for Xan been shown to have a broad substrate speci®city including Hx, measured by enzymatic parameters was low relative to that for U, Xan, AP site and mismatched bases (7,21,46,47). its physiological substrate (Tg) (Table 2). However, the low In the present work, it has been shown that the alkA nei but signi®cant activity may be crucial for repair of Xan when double mutant, but not the alkA and nei single mutants, was the level of AlkA is not suf®ciently high, e.g. in uninduced sensitive to nitrous acid (Fig. 8). These results suggest a cells. Endo VIII was originally found as a repair enzyme for degenerative role of AlkA and Endo VIII in the repair of a Tg and urea residues (31), and later it was shown to recognize certain class of nitrous acid-induced DNA lesions, for example other oxidative pyrimidine damage such as 5-hydroxycyto- Xan and Oxa, though involvement of other E.coli base sine, 5-hydroxyuracil and uracil glycol (33,42). Thus, sub- excision repair enzymes such as MutY, Mug and TagI has not strate speci®city of Endo VIII is similar to that of Endo III. been eliminated in this study. The actual mechanisms However, the amino acid sequence of Endo VIII shows no operating in E.coli cells to counteract the lethal and mutagenic homology to that of Endo III. Rather, the N-terminal and effects of nitrous acid and NO appear to be rather complicated. C-terminal regions of Endo VIII are similar to those of Fpg Several studies show cell killing and mutagenicity by nitrous involved in repair of oxidative purine lesions in E.coli (32). acid or NO increase in the cells de®cient in nucleotide Furthermore, Endo VIII has been shown recently to excise excision repair (48±50) or recombination repair (51). In oxidative purine lesions including 7,8-dihydro-8-oxoguanine contrast, studies on n® (the Endo V gene) mutants by Weiss's and formamidopyrimidine (25,43). Thus, Endo VIII has a group indicate the involvement of Endo V in avoidance of potential capacity to accept purine lesions as well as nitrous acid-induced mutations (52±54). Thus, at least four pyrimidine lesions, though the activities may vary. In this repair pathways including base excision repair indicated by context, it may not be surprising that Endo VIII recognized this study, nucleotide excision repair, recombination repair, Xan and Oxa. However, questions remain as to why Xan was a and repair initiated by Endo V have been implicated in better substrate than Oxa for Endo VIII and why Xan and Oxa restoration of DNA lesions formed by nitrous acid and NO. It were substrates for Endo VIII but not for Fpg. Concerning the is not clear whether such differences originate from dosage or latter, the three-dimensional structures of Endo VIII and Fpg types of nitrogen oxide species, cell conditions or accessed Downloaded from https://academic.oup.com/nar/article-abstract/30/22/4975/2380497 by Ed 'DeepDyve' Gillespie user on 31 January 2018 Nucleic Acids Research, 2002, Vol. 30 No. 22 4983 19. Suzuki,T., Yoshida,M., Yamada,M., Ide,H., Kobayashi,M., Kanaori,K., phenotypes (lethal or mutation effect). Thus, further studies Tajima,K. and Makino,K. (1998) Misincorporation of 2¢-deoxyoxanosine are necessary to clarify the whole picture of the cellular 5¢-triphosphate by DNA polymerases and its implication for mutagenesis. mechanisms to counteract mutagenic and lethal effects Biochemistry, 37, 11592±11598. associated with nitrogen oxide species. 20. Suzuki,T., Yamada,M., Ide,H., Kanaori,K., Tajima,K., Morii,T. and Makino,K. (2000) Identi®cation and characterization of a reaction product of 2¢-deoxyoxanosine with glycine. Chem. Res. Toxicol., 13, ACKNOWLEDGEMENTS 227±230. 21. He,B., Qing,H. and Kow,Y.W. (2000) Deoxyxanthosine in DNA is This research was supported by Grants-in-Aid from the repaired by Escherichia coli endonuclease V. Mutat. Res., 459, 109±144. Ministry of Education, Culture, Sports, Science and 22. Wang,G., Palejwala,V.A., Dunman,P.M., Aviv,D.H., Murphy,H.S., Rahman,M.S. and Humayun,M.Z. (1995) Alkylating agents induce Technology, Japan (to H.I. and K.M.). UVM, a recA-independent inducible mutagenic phenomenon in Escherichia coli. Genetics, 141, 813±823. 23. Saito,Y., Uraki,F., Nakajima,S., Asaeda,A., Ono,K., Kubo,K. and REFERENCES Yamamoto,K. (1997) Characterization of endonuclease III (nth) and 1. Frederico,L.A., Kunkel,T.A. and Shaw,B.R. (1990) A sensitive genetic endonuclease VIII (nei) mutants of Escherichia coli K-12. J. Bacteriol., assay for the detection of cytosine deamination: determination of rate 179, 3783±3785. constants and the activation energy. Biochemistry, 29, 2532±2537. 24. Masaoka,A., Terato,H., Kobayashi,M., Honsho,A., Ohyama,Y. and 2. Lindahl,T. (1993) Instability and decay of the primary structure of DNA. Ide,H. (1999) Enzymatic repair of 5-formyluracil. I. Excision of 5- Nature, 362, 709±715. formyluracil site-speci®cally incorporated into oligonucleotide substrates 3. Duncan,B.K. and Weiss,B. (1982) Speci®c mutator effects of ung (uracil- by AlkA protein (Escherichia coli 3-methyladenine DNA glycosylase II). DNA glycosylase) mutations in Escherichia coli. J. Bacteriol., 151, J. Biol. Chem., 274, 25136±25143. 750±755. 25. Asagoshi,K., Yamada,T., Okada,Y., Terato,H., Ohyama,Y., Seki,S. and 4. Hill-Perkins,M., Jones,M.D. and Karran,P. (1986) Site-speci®c Ide,H. (2000) Recognition of formamidopyrimidine by Escherichia coli mutagenesis in vivo by single methylated or deaminated purine bases. and mammalian thymine glycol glycosylases. J. Biol. Chem., 275, Mutat. Res., 162, 153±163. 24781±24786. 5. Friedberg,E.C., Walker,G.C. and Siede,W. (1995) DNA Repair and 26. Sarker,A.H., Ikeda,S., Nakano,H., Terato,H., Ide,H., Imai,K., Mutagenesis. ASM Press, Washington, DC. Akiyama,K., Tsutsui,K., Bo,Z., Kubo,K., Yamamoto,K., Yasui,A., 6. Saparbaev,M. and Laval,J. (1994) Excision of hypoxanthine from DNA Yoshida,M.C. and Seki,S. (1998) Cloning and characterization of a containing dIMP residues by the Escherichia coli, yeast, rat and human mouse homologue (mNthl1) of Escherichia coli endonuclease III. alkylpurine DNA glycosylases. Proc. Natl Acad. Sci. USA, 91, J. Mol. Biol., 282, 761±774. 5873±5877. 27. Asagoshi,K., Odawara,H., Nakano,H., Miyano,T., Terato,H., Ohyama,Y., 7. Yao,M., Hatahet,Z., Melamede,R.J. and Kow,Y.W. (1994) Puri®cation Seki,S. and Ide,H. (2000) Comparison of substrate speci®cities of and characterization of a novel deoxyinosine-speci®c enzyme, Escherichia coli endonuclease III and its mouse homologue (mNTH1) deoxyinosine 3¢ endonuclease, from Escherichia coli. J. Biol. Chem., using de®ned oligonucleotide substrates. Biochemistry, 39, 269, 16260±16268. 11389±11398. 8. Lindahl,T. (1979) DNA glycosylases, endonuclease for apurinic/ 28. Ide,H., Okagami,M., Murayama,H., Kimura,Y. and Makino,K. (1993) apyrimidinic sites and base excision repair. Prog. Nucleic Acid Res. Mol. Synthesis and characterization of oligonucleotides containing the alpha- Biol., 22, 135±192. anomer of deoxyadenosine to study its in¯uence on DNA replication. 9. Marletta,M.A. (1989) Nitric oxide: biosynthesis and biological Biochem. Mol. Biol. Int., 31, 485±491. signi®cance. Trends Biochem. Sci., 14, 488±492. 29. Miller,J.H. (1972) Experiments in Molecular Genetics. Cold Spring 10. Ohshima,H. and Bartsch,H. (1994) Chronic infections and in¯ammatory Harbor Laboratory Press, Cold Spring Harbor, NY. processes as cancer risk factors: possible role of nitric oxide in 30. Thomas,L., Yang,C.-H. and Goldthwait,D.A. (1982) Two DNA carcinogenesis. Mutat. Res., 305, 253±264. glycosylases in Escherichia coli which release primarily 3- 11. Wink,D.A., Vodovots,Y., Laval,J., Laval,F., Dewhirst,M.W. and methyladenine. Biochemistry, 21, 1162±1169. Mitchell,J.B. (1998) The multifaceted roles of nitric oxide in cancer. 31. Melamede,R.J., Hatahet,Z., Kow,Y.W., Ide,H. and Wallace,S.S. (1994) Carcinogenesis, 19, 711±721. Isolation and characterization of endonuclease VIII from Escherichia 12. Wink,D.A., Kasprzak,K.S., Maragos,C.M., Elespuru,R.K., Misra,M., coli. Biochemistry, 33, 1255±1264. Dunams,T.M., Cebula,T.A., Koch,W.H., Andrews,A.W., Allen,J.S. and 32. Jiang,D., Hatahet,Z., Blaisdell,J.O., Melamede,R.J. and Wallace,S.S. Keefer,L.K. (1991) DNA deaminating ability and genotoxicity of nitric (1997) Escherichia coli endonuclease VIII: cloning, sequencing, and oxide and its progenitors. Science, 254, 1001±1003. overexpression of the nei structural gene and characterization of nei and 13. Suzuki,T., Yamaoka,R., Nishi,M., Ide,H. and Makino,K. (1996) Isolation nei nth mutants. J. Bacteriol., 179, 3773±3782. and characterization of a novel product, 2¢-deoxyoxanosine, from 2¢- 33. Jiang,D., Hatahet,Z., Melamede,R.J., Kow,Y.W. and Wallace S.S. (1997) deoxyguanosine, oligodeoxynucleotide, and calf thymus DNA treated by Characterization of Escherichia coli endonuclease VIII. J. Biol. Chem., nitrous acid and nitric oxide. J. Am. Chem. Soc., 118, 2515±2516. 272, 32230±32239. 14. Lucas,L.T., Gatehouse,D. and Shuker,D.E. (1999) Ef®cient nitroso group 34. Lindahl,T., Sedgwick,B., Sekiguchi,M. and Nakabeppu,Y. (1988) transfer from N-nitrosoindoles to nucleotides and 2¢-deoxyguanosine at Regulation and expression of the adaptive response to alkylating agents. physiological pH. J. Biol. Chem., 274, 18319±18326. Annu. Rev. Biochem., 57, 133±157. 15. Suzuki,T., Ide,H., Yamada,M., Endo,N., Kanaori,K., Tajima,K., Morii,T. 35. Bessman,M.J., Lehman,I.R., Adler,J., Zimmerman,S.B., Simms,E.S. and and Makino,K. (2000) Formation of 2¢-deoxyoxanosine from 2¢- Kornberg,A. (1958) Enzymatic synthesis of deoxyribonucleic acid. III. deoxyguanosine and nitrous acid: mechanism and intermediates. Nucleic The incorporation of pyrimidine and purine analogues into Acids Res., 28, 544±551. deoxyribonucleic acid. Proc. Natl Acad. Sci. USA., 44, 633±640. 16. Suzuki,T., Matsumura,Y., Ide,H., Kanaori,K. Tajima,K. and Makino,K. 36. Bjelland,S., Birkeland,N.K., Benneche,T., Volden,G. and Seeberg,E. (1997) Deglycosylation susceptibility and base-pairing stability of 2¢- (1994) DNA glycosylase activities for thymine residues oxidized in the deoxyoxanosine in oligonucleotide. Biochemistry, 36, 8013±8019. methyl group are functions of the AlkA enzyme in Escherichia coli. 17. Eritja,R. Horowitz,D.M., Walker,P.A., Ziehler-Martin,J.P., J. Biol. Chem., 269, 30489±30495. Boosalis,M.S., Goodman,M.F., Itakura,K. and Kaplan,B.E. (1986) 37. Terato,H., Masaoka,A., Kobayashi,M., Fukushima,S., Ohyama,Y., Synthesis and properties of oligonucleotides containing 2¢- Yoshida,M. and Ide,H. (1999) Enzymatic repair of 5-formyluracil II. deoxynebularine and 2¢-deoxyxanthosine. Nucleic Acids Res., 24, Mismatch formation between 5-formyluracil and guanine during DNA 168±175. replication and its recognition by two proteins involved in base excision 18. Kamiya,H., Shimizu,M. Inoue,H. and Ohtsuka,E. (1992) Mutation induced by deoxyxanthosine in codon 12 of a synthetic c-Ha-ras gene. repair (AlkA) and mismatch repair (MutS). J. Biol. Chem., 274, Nucl. Nucl., 11, 247±260. 25144±25150. Downloaded from https://academic.oup.com/nar/article-abstract/30/22/4975/2380497 by Ed 'DeepDyve' Gillespie user on 31 January 2018 4984 Nucleic Acids Research, 2002, Vol. 30 No. 22 38. Privezentzev,C.V., Saparbaev,M., Sambandam,A., Greenberg,M.M. and 46. Yao,M. and Kow,Y.W. (1995) Interaction of deoxyinosine 3¢- Laval,J. (2000) AlkA protein is the third Escherichia coli DNA repair endonuclease from Escherichia coli with DNA containing deoxyinosine. protein excising a ring fragmentation product of thymine. Biochemistry, J. Biol. Chem., 270, 28609±28616. 39, 14263±14268. 47. Yao,M. and Kow,Y.W. (1997) Further characterization of Escherichia 39. Yamagata,Y., Kato,M., Odawara,K., Tokuno,Y., Nakashima,Y., coli endonuclease V. J. Biol. Chem., 272, 30774±30779. Tatsushima,N., Yasumura,K., Tomita,K., Ihara,K., Fujii,Y., 48. Hartman,Z., Henrickson,E.N., Hartman,P.E. and Cebula,T.A. (1994) Nakabeppu,Y., Sekiguchi,M. and Fujii,S. (1996) Three dimensional Molecular models that may account for nitrous acid mutagenesis in structure of a DNA repair enzyme, 3-methyladenine DNA glycosylase II, organisms containing double-stranded DNA. Environ. Mol. Mutagen., from Escherichia coli. Cell, 86, 311±319. 24, 168±175. 40. Labahn,J., Scharer,O.D., Long,A., Ezaz-Nikpay,K., Verdine,G.L. and 49. Sidorkina,O., Saparbaev,M. and Laval,J. (1997) Effects of nitrous acid Ellenberger,T.E. (1996) Structure basis for the excision repair of treatment on the survival and mutagenesis of Escherichia coli cells alkylation-damaged DNA. Cell, 86, 321±329. lacking base excision repair (hypoxanthine-DNA glycosylase-ALK A 41. Hollis,T., Ichikawa,Y. and Ellenberger,T. (2000) DNA bending and a protein) and/or nucleotide excision repair. Mutagenesis, 12, 23±28. ¯ip-out mechanism for base excision by the helix±hairpin±helix DNA 50. Tamir,S., Burney,S. and Tannenbaum,S.R. (1996) DNA damage by nitric glycosylase, Escherichia coli AlkA. EMBO J., 19, 758±766. oxide. Chem. Res. Toxicol., 9, 821±827. 42. Purmal,A.A., Lampman,G.W., Bond,J.P., Hatahet,Z. and Wallace S.S. 51. Spek,E.J., Wright,T.L., Stitt,M.S., Taghizadeh,N.R., Tannenbaum,S.R., (1998) Enzymatic processing of uracil glycol, a major oxidative product Marinus,M.G. and Engelward,B.P. (2001) Recombinational repair is of DNA cytosine. J. Biol. Chem., 273, 10026±10035. critical for survival of Escherichia coli exposed to nitric oxide. 43. Blaisdell,J.O., Hatahet,Z. and Wallace,S.S. (1999) A novel role for J. Bacteriol., 183, 131±138. Escherichia coli endonuclease VIII in prevention of spontaneous G®T 52. Guo,G. and Weiss,B. (1998) Endonuclease V (n®) mutant of Escherichia transversions. J. Bacteriol., 181, 6396±6402. coli K-12. J. Bacteriol., 180, 46±51. 44. Zharkov,D.O., Golan,G., Gilboa,R., Fernandes,A.S., Gerchman,S.E., 53. Schouten,K.A. and Weiss,B. (1999) Endonuclease V protects Kycia,J.H., Rieger,R.A., Grollman,A.P. and Shoham,G. (2002) Structural Escherichia coli against speci®c mutations caused by nitrous acid. analysis of an Escherichia coli endonuclease VIII covalent reaction Mutat. Res., 435, 245±254. intermediate. EMBO J., 21, 789±800. 54. Weiss,B. (2001) Endonuclease V of Escherichia coli prevents mutations 45. Gilboa,R., Zharkov,D.O., Golan,G., Fernandes,A.S., Gerchman,S.E., from nitrosative deamination during nitrate/nitrite respiration. Matz,E., Kycia,J.H., Grollman,A.P. and Shoham,G. (2002) Structure of Mutat. Res., 461, 301±309. formamidopyrimidine-DNA glycosylase covalently complexed to DNA. J. Biol. Chem., 277, 19811±19816. Downloaded from https://academic.oup.com/nar/article-abstract/30/22/4975/2380497 by Ed 'DeepDyve' Gillespie user on 31 January 2018 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Nucleic Acids Research Oxford University Press

Novel repair activities of AlkA (3‐methyladenine DNA glycosylase II) and endonuclease VIII for xanthine and oxanine, guanine lesions induced by nitric oxide and nitrous acid

Loading next page...
 
/lp/oxford-university-press/novel-repair-activities-of-alka-3-methyladenine-dna-glycosylase-ii-and-3RogFzXHvG

References (55)

Publisher
Oxford University Press
ISSN
0305-1048
eISSN
1362-4962
DOI
10.1093/nar/gkf630
Publisher site
See Article on Publisher Site

Abstract

ã 2002 Oxford University Press Nucleic Acids Research, 2002, Vol. 30 No. 22 4975±4984 Novel repair activities of AlkA (3-methyladenine DNA glycosylase II) and endonuclease VIII for xanthine and oxanine, guanine lesions induced by nitric oxide and nitrous acid Hiroaki Terato, Aya Masaoka, Kenjiro Asagoshi, Akiko Honsho, Yoshihiko Ohyama, 1 1 1 2 Toshinori Suzuki , Masaki Yamada , Keisuke Makino , Kazuo Yamamoto and Hiroshi Ide* Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Higashi-Hiroshima 739-8526, Japan, Institute of Advanced Energy, Kyoto University, Gokasho, Uji 611-0011, Japan and Biological Institute, Graduate School of Science, Tohoku University, Sendai 980-8578, Japan Received May 21, 2002; Revised July 18, 2002; Accepted September 24, 2002 ABSTRACT that approximately 100 C residues are deaminated per human cell per day (2). If unrepaired the resulting uracil (U) induces Nitrosation of guanine in DNA by nitrogen oxides CG®TA transitions (3). Deamination of A gives rise to such as nitric oxide (NO) and nitrous acid leads to hypoxanthine (Hx), which pairs with C and induces AT®GC formation of xanthine (Xan) and oxanine (Oxa), transitions (4). Since U and Hx are mutagenic, organisms are potentially cytotoxic and mutagenic lesions. In the equipped with repair mechanisms for these lesions. Uracil in present study, we have examined the repair capacity DNA is removed by uracil-DNA glycosylase, which is highly of DNA N-glycosylases from Escherichia coli for conserved in prokaryotic and eukaryotic organisms (5). Xan and Oxa. The nicking assay with the de®ned Hypoxanthine is generally excised from DNA by a family of methyl purine DNA glycosylases (6) and Escherichia coli has substrates containing Xan and Oxa revealed that an extra enzyme for Hx, namely endonuclease (Endo) V, AlkA [in combination with endonuclease (Endo) IV] which incises the second phosphodiester bond on the 3¢ side of and Endo VIII recognized Xan in the tested enzymes. Hx (7). The activity (V /K ) of AlkA for Xan was 5-fold max m Guanine undergoes spontaneous hydrolytic deamination to lower than that for 7-methylguanine, and that of yield xanthine (Xan), but the spontaneous deamination rate of Endo VIII was 50-fold lower than that for thymine G is lower than that of C and A (8). Therefore, so far as glycol. The activity of AlkA and Endo VIII for Xan spontaneous deamination is concerned, deamination of G is was further substantiated by the release of [ H]Xan biologically less important than that of C or A. However, from the substrate. The treatment of E.coli with nitrogen oxides such as nitric oxide (NO) and nitrous acid N-methyl-N ¢-nitro-N-nitrosoguanidine increased the (HNO ) induce deamination of DNA bases with signi®cant Xan-excising activity in the cell extract from alkA rates. In contrast to spontaneous hydrolytic deamination, G is but not alkA strains. The alkA and nei (the Endo VIII the most sensitive of the three bases to nitric oxide and nitrous gene) double mutant, but not the single mutants, acid. NO has been characterized primarily as a second messenger exerting various physiological activities (9). In exhibited increased sensitivity to nitrous acid rela- humans, 1 mmol of NO is constitutively generated per body tive to the wild type strain. AlkA and Endo VIII also per day and the amount increases by 10±100 times upon exhibited excision activity for Oxa, but the activity bacterial infection or in¯ammation. NO overproduced by was much lower than that for Xan. activated macrophages in chronically in¯amed tissues has been implicated as carcinogenic by virtue of its ability to cause DNA damage (10,11). Nitrous anhydride (N O ), formed by 2 3 INTRODUCTION autoxidation of NO, is a powerful nitrosating agent, and induces deamination of C, A and G in nucleosides and DNA, DNA that stores genetic information of cells shows certain structural instability. The DNA bases bearing an exocyclic generating U, Hx and Xan, respectively (12). Nitrous acid that amino group [adenine (A), guanine (G), cytosine (C)] undergo is formed by protonation of sodium nitrite (NaNO ), present in deamination. The rate of hydrolytic deamination is the highest food intake, also induces base deamination via N O . 2 3 for C under physiological conditions (1), and it is estimated Although nitrosation of C and A leads exclusively to U and *To whom correspondence should be addressed. Tel/Fax: +81 824 24 7457; Email: ideh@hiroshima-u.ac.jp Present address: Toshinori Suzuki, Unit of Endogenous Cancer Risk Factors, International Agency for Research on Cancer, Lyon, France Downloaded from https://academic.oup.com/nar/article-abstract/30/22/4975/2380497 by Ed 'DeepDyve' Gillespie user on 31 January 2018 4976 Nucleic Acids Research, 2002, Vol. 30 No. 22 Table 1. List of oligonucleotides used in this study Abbreviation Sequence 19T 5¢-ACAGACGCCATCAACCAGG 19TG 5¢-ACAGACGCCATgCAACCAGG COM19A 3¢-TGTCTGCGGTAGTTGGTCC PRIM15 5¢-CATCGATAGCATCCT Figure 1. Products formed by the reaction of guanine (G) with nitrogen 25G 5¢-CATCGATAGCATCCTGCCTTCTCTC oxides (NO and HNO ). 25MG 5¢-CATCGATAGCATCCTMCCTTCTCTC 25XAN 5¢-CATCGATAGCATCCTXCCTTCTCTC 25OXA 5¢-CATCGATAGCATCCTOCCTTCTCTC COM25A 3¢-GTAGCTATCGTAGGAAGGAAGAGAG Hx, respectively, that of G gives rise to not only Xan but also COM25T 3¢-GTAGCTATCGTAGGATGGAAGAGAG oxanine (Oxa) with a molar ratio of 3 (Xan):1 (Oxa) (Fig. 1) COM25G 3¢-GTAGCTATCGTAGGAGGGAAGAGAG (13). Recently, it has been shown that Xan and Oxa are formed COM25C 3¢-GTAGCTATCGTAGGACGGAAGAGAG COM30C 3¢-AAGTCGTAGCTATCGTAGGACGGAAGAGAG by nitroso group transfer to G from N-nitrosoindoles (14). Xan and Oxa are produced via the common precursor (a diazoate Tg, thymine glycol; M, 7-methylguanine; X, xanthine; O, oxanine. Base derivative of the 2-amino group of G), but formation of Oxa lesions and paired bases are underlined. involves deamination of the 2-amino group followed by rearrangement of the ring atoms (15). The biochemical and genotoxic effects of Xan and Oxa have been assessed nei alkA phenotypes were con®rmed by measuring Endo VIII previously by several studies. Substitution of Xan or Oxa for and Endo VIII/AlkA activities and the strains were designated G in duplex DNA results in large decreases in the helix KY100 (MV1161 + nei) and KY101 (MV1571 + nei), stability (16). Xan in template DNA directs incorporation of T respectively. Endo III, Endo VIII, Endo IV, formamido- as well as C during DNA replication (17,18). Similarly, pyrimidine DNA glycosylase (Fpg/MutM) and AlkA were 2¢-deoxyribonucleoside 5¢-triphosphates of Xan (dXTP) and puri®ed from E.coli strains that overproduced these enzymes Oxa (dOTP) are incorporated opposite template T as well as C (24,25). Escherichia coli uracil DNA glycosylase (Ung) and by DNA polymerase, though their incorporation ef®ciencies exonuclease (Exo) III were purchased from New England are much lower than dGTP (19). In addition, Xan but not Oxa Biolabs. Escherichia coli DNA polymerase I Klenow frag- can be spontaneously converted to a cytotoxic and mutagenic ment (Pol I Kf) and T4 polynucleotide kinase were obtained AP (apurinic/apyrimidinic) site due to its labile N-glycosidic from Life Technologies. 2¢-Deoxycytidine 5¢-triphosphate bond (16). Thus, Xan may show additional biological effects (dCTP), thymidine 5¢-triphosphate (dTTP) and [g- P]adeno- after conversion to an AP site. Recently, Oxa has been sine 5¢-triphosphate (ATP) (110 TBq/mmol) were from shown to form a covalently bound adduct with glycine, Amersham Biosciences. 2¢-[8- H]Deoxyguanosine 5¢-triphos- suggesting a novel cytotoxic/genotoxic mechanism for this phate (dGTP) (37 MBq/244 pmol) was purchased from NEN lesion (20). These data combined together indicate the Life Science Products. Guanine and Xan were purchased from necessity of repair capacities for Xan and Oxa in cells. Sigma. However, studies on the enzymatic repair of these lesions are extremely limited (21). Oligonucleotides In the present work, we have examined the repair capacity Oligonucleotides without modi®ed bases were purchased from of E.coli base excision repair enzymes for Xan and Oxa using Espec Oligo Service and puri®ed by reversed phase HPLC. de®ned oligonucleotide substrates containing these lesions. Preparation of 25MG and 19TG which contain a single We report here that, among the enzymes tested, AlkA and 7-methylguanine (7mG) and thymine glycol (Tg), respect- Endo VIII exhibit repair activities for Xan and Oxa in a paired ively, were reported previously (26,27). The sequences of the base-dependent manner and the repair ef®ciency for Oxa is oligonucleotides used in this study are shown in Table 1. much lower than for Xan. Induction of the alkA gene encoding AlkA protein in E.coli cells results in an increased releasing Preparation of dXTP and dOTP and their incorporation activity of Xan. Furthermore, E.coli de®cient in both AlkA into oligonucleotides and Endo VIII, but not either of them alone, exhibits increased sensitivity to nitrous acid. 2¢-Deoxyxanthosine 5¢-triphosphate (dXTP) and 2¢-deoxy- oxanosine 5¢-triphosphate (dOTP) were synthesized from dGTP. dGTP (1 mM) was incubated with 100 mM NaNO at 37°C in 3 M acetate buffer (pH 3.7) for 2 h, and the resulting MATERIALS AND METHODS dXTP and dOTP were puri®ed by reversed phase HPLC. The Strains, enzymes and chemicals detailed procedures and characterization of dXTP and dOTP Escherichia coli MV1161 (thr1, ara14, leuB6, D(gpt-proA)62, were reported previously (19). [ H]dXTP was prepared 3 3 lacY1, tsx33, supE44, galK2, hisG4, rfbD1, mgl51, rpsL31, from [ H]dGTP in an essentially similar manner. [ H]dGTP kdgK51, xyl5, mtl1, argE3, thi1, rfa550) and MV1571 (37 MBq/244 nmol, diluted with cold dGTP) was incubated [alkA51::Mud1 (Amp lac) in MV1161] were laboratory stocks with 100 mM NaNO in 1 M acetate buffer (pH 3.7, 100 ml) at (22). To construct Endo VIII mutants, MV1161 and MV1571 37°C for 2 h. [ H]dXTP was puri®ed by reversed phase HPLC. were infected with P1 phage carrying the Dnei::Km allele Oligonucleotide substrates containing Xan and Oxa were derived from NKJ1003 (23) and were selected for the prepared by DNA polymerase reactions. PRIM15 was 5¢-end kanamycin (Km)-resistant phenotype. The resultant nei and labeled using [g- P]ATP and T4 polynucleotide kinase and Downloaded from https://academic.oup.com/nar/article-abstract/30/22/4975/2380497 by Ed 'DeepDyve' Gillespie user on 31 January 2018 Nucleic Acids Research, 2002, Vol. 30 No. 22 4977 puri®ed by a Sep-pak cartridge (Waters) (28). PRIM15 (100 fmol) were incubated with AlkA (600 fmol) and Ung (50 pmol) and COM25C (100 pmol, 2-fold molar excess) in (170 mU) in buffer D (10 ml) and buffer E (10 ml), respectively, 10 mM Tris±HCl (pH 7.5) and 25 mM NaCl were heated at at 37°C for 30 min. The composition of buffer D was 50 mM 90°C for 10 min and then annealed at room temperature. HEPES-KOH (pH 7.5), 1 mM EDTA and 5 mM 2-mercapto- PRIM15/COM25C (25 pmol) in the polymerase buffer ethanol and that of buffer E was 20 mM Tris±HCl (pH 8.0), (200 ml) were incubated with dXTP or dOTP (both 20 nmol) 1 mM EDTA and 1 mM dithiothreitol. After incubation, the and Pol I Kf (25 U) at 25°C for 5 min to introduce a single substrates were puri®ed by phenol extraction and ethanol residue of Xan or Oxa opposite C (16th position from the 3¢ precipitation. The puri®ed substrates were incubated further terminus in COM25C). The polymerase buffer consisted of with Endo IV (120 fmol) at 37°C for 30 min to incise abasic 66 mM Tris±HCl (pH 7.0), 1.5 mM 2-mercaptoethanol and sites. The sample was mixed with gel loading buffer and 6.6 mM MgCl . Then dCTP and dTTP (10 nmol each) were separated by 16% denaturing PAGE. The radioactivity of added to the reaction mixture to extend the primer fully. The products was analyzed on a BAS2000 phosphorimaging oligonucleotides containing Xan and Oxa were designated analyzer (Fuji Film). Paired base effects on the activity of 25XAN and 25OXA, respectively. The duplexes (25XAN/ AlkA and Endo VIII for Xan and Oxa were measured using COM25C and 25OXA/COM25C) were puri®ed by phenol 25XAN/COM25N and 25OXA/COM25N (N = A, G, C or T) extraction, ethanol precipitation and gel ®ltration on a as substrates. The reactions were performed in a manner Sephadex G-25 column (1 ml). The site-speci®c introduction essentially similar to those described above. The amounts of of Xan and Oxa into 25XAN and 25OXA, respectively, was the substrates (25XAN and 25OXA) and the enzymes (AlkA con®rmed by the heat/acid and ammonia/piperidine treat- and Endo VIII) were 100 fmol and 300 fmol±3 pmol, respectively. ments (see Results). For preparation of oligonucleotide substrates containing Xan:N and Oxa:N pairs (N = A, G, C, For analysis of the kinetic parameters of AlkA, 25XAN/ T), the DNA polymerase reaction was performed in a similar COM25C and 25MG/COM25C (50±1000 fmol) were incu- manner except that COM30C was used as a template in place bated with 300 fmol of AlkA at 37°C for 10 min in buffer D of COM25C. This facilitated the subsequent electrophoretic (10 ml), and then the products were treated with Endo IV as separation of 25XAN and 25OXA from the template described above. For Endo VIII, 25XAN/COM25C and 19TG/ (COM30C). After the reaction, 25XAN and 25OXA were COM19A (50±1000 fmol) were incubated with Endo VIII puri®ed by 16% denaturing polyacrylamide gel electro- (300 fmol) at 37°C for 15 min (25XAN/COM25C) and 2 min phoresis (PAGE), extracted from the gel and desalted by a (19TG) in buffer A (10 ml). Reaction products were separated Sep-pak cartridge. Puri®ed 25XAN and 25OXA were and quanti®ed as described above. The parameters V and max annealed with COM25N (N = A, G, C, T). 25XAN containing K were evaluated from Michaelis±Menten plots using a [ H]Xan was prepared as described above using PRIM15/ hyperbolic curve ®tting program. COM25C, [ H]dXTP and Pol I Kf for the initial polymeriza- Release assays of Xan with AlkA and Endo VIII 3 3 tion reaction. 25G containing [ H]G in place of [ H]Xan was also prepared using [ H]dGTP. To measure the N-glycosylase activity of AlkA, 25XAN/ COM25C and 25G/COM25C (both 2.25 pmol) containing Chemical cleavage reactions of oligonucleotides 3 3 [ H]Xan and [ H]G, respectively, were incubated with AlkA 25XAN, 25OXA and 25G (all P-labeled at the 5¢ end, (3 pmol) at 37°C for 30 min in buffer D (20 ml). The reaction 100 fmol) were heated at 90°C for 30 min under acidic with Endo VIII (6 pmol) was performed in a similar manner conditions (pH 3.5, adjusted by acetic acid) and the solution using buffer A. After the reaction, the sample was loaded onto was evaporated to dryness. Alternatively, the oligonucleotides a Sephadex G-25 ®ne gel ®ltration column (f 3 3 300 mm) to were treated ®rst by 30% ammonia at 65°C for 4 h. After separate the released product and the oligonucleotides. The removing ammonia by evaporation, the samples were treated column was eluted by water and the eluent was collected every by 10% piperidine at 90°C for 30 min and evaporated to 100 ml. Aliquots of the collected fractions were subjected to dryness. The products from both treatments were mixed with liquid scintillation counting. The oligonucleotides and the gel loading buffer [95% (v/v) formamide, 20 mM EDTA, released product were eluted in fractions 12±15 and 20±25, 0.1% (w/v) xylene cyanol and 0.1% (w/v) bromophenol blue] respectively, under these conditions. The pooled fractions and analyzed by 16% denaturing PAGE. containing the released product were evaporated to dryness and resuspended in a small volume of water. The sample was Nicking assays for the activity to Xan and Oxa analyzed by reversed phase HPLC. The HPLC system In the reaction with Fpg, Endo III and Endo VIII (all consisted of Hitachi L-6200 pumps equipped with a reversed N-glycosylase/AP lyase), 25G/COM25C, 25XAN/COM25C phase WS-DNA column (f 4.6 3 150 mm, Wako) and a and 25OXA/COM25C (100 fmol) were incubated with the Hitachi L-4200H UV-VIS detector. Elution was carried out enzyme (600 fmol) in buffer A (10 ml) at 37°C for 30 min. with a linear gradient of acetonitrile (0±20%, v/v) in 20 mM Buffer A comprised 10 mM Tris±HCl (pH 7.5), 1 mM EDTA sodium phosphate buffer (pH 5.0) at a ¯ow rate 0.8 ml/min. and 100 mM NaCl. The reactions with Endo IV (120 fmol) and The column eluent was collected every 20 s and was subjected Exo III (0.1 U) (both AP endonucleases) were similarly to liquid scintillation counting. carried out in buffer B (10 ml) and C (10 ml), respectively. The Release assays of Xan with crude cell extracts composition of buffer B was 10 mM Tris±HCl (pH 7.5), 1 mM EDTA and 50 mM NaCl and that of buffer C was 10 mM Escherichia coli MV1161 (wild type) and MV1571 (alkA) Tris±HCl (pH 7.5), 2 mM CaCl and 1 mM EDTA. In the were cultivated in LB media (250 ml) in the absence reaction with simple DNA glycosylases, the substrates (MV1161) and presence (MV1571) of 50 mg/ml ampicillin Downloaded from https://academic.oup.com/nar/article-abstract/30/22/4975/2380497 by Ed 'DeepDyve' Gillespie user on 31 January 2018 4978 Nucleic Acids Research, 2002, Vol. 30 No. 22 at 37°C for 3 h under aeration. Depending on the experiments, N-methyl-N ¢-nitro-N-nitrosoguanidine (MNNG) (®nal con- centration 20 mM) was added into the culture media and incubation was continued for further 1.5 h. After recovering cells by centrifugation, the cell pellet was suspended in 10 ml of 300 mM Tris±HCl (pH 8.0), 5 mM EDTA and 1 mg/ml lysozyme, and was lyzed by repeated freeze/thaw cycles. The lyzed cells were centrifuged at 43 000 g for 1 h at 4°C and the supernatant was recovered. The proteins in the supernatant were collected by ammonium sulfate precipitation (60% saturation) and were resuspended in 10 mM Tris±HCl (pH 7.5) and 1 mM EDTA (TE buffer). After dialysis against TE buffer at 4°C, the cell extract was used for the release assay of [ H]Xan. The protein concentration was determined by a BCA protein assay reagent (Pierce) using BSA as a standard. The release assay was performed as described for AlkA using 5 mg of protein. Figure 2. Chemical cleavage of oligonucleotides containing G (25G), Xan Nitrous acid sensitivity of alkA and nei E.coli strains (25XAN) and Oxa (25OXA). 5¢- P-labeled 25G, 25XAN and 25OXA were A modi®ed procedure by Miller (29) was used. Escherichia heated at 90°C for 30 min in an acidic aqueous solution (pH 3.5, adjusted coli strains, MV1161 (wild type), MV1571 (alkA), KY100 by acetic acid) (lanes 3, 6 and 9). Alternatively they were incubated ®rst in 30% (v/v) ammonia at 65°C for 4 h, and then in 10% (v/v) piperidine at (nei) and KY101 (alkA nei) were cultured overnight in 1 ml of 90°C for 30 min as described in Materials and Methods (lanes 4, 7 and 10). LB media with 50 mg/ml of appropriate antibiotics (ampicillin The samples were separated by 16% denaturing PAGE. Lane 1 shows a 5¢- for MV1571, kanamycin for KY100, and ampicillin and P-labeled marker (PRIM15). The bands of b-elimination, d-elimination kanamycin for KY101) at 37°C. These cultures were diluted and 3¢-OH products are indicated by arrows. into 100 ml of LB media with or without antibiotics and cells were grown to a logarithmic phase (OD = 0.6) at 37°C. After washing in acetate buffer (0.1 M, pH 4.6), cells were described in Materials and Methods. PAGE analysis of the incubated in NaNO (0±30 mM) in 5 ml of acetate buffer treated oligonucleotides showed that 25XAN was speci®cally (0.1 M, pH 4.6) at 37°C for 10 min. To stop incubation, an cleaved by the heat/acid treatment (Fig. 2, lane 9) but not the equal volume of minimal A medium [60.3 mM K HPO , 2 4 ammonia/piperidine treatment (Fig. 2, lane 10). According to 33.1 mM KH PO , 7.6 mM (NH ) SO and 1.7 mM trisodium 2 4 4 2 4 the gel mobility of the products, the cleaved products were citrate] was added. After washing in minimal A medium 15mers with different 3¢ terminal modi®cations resulting twice, the cell suspension was appropriately diluted and from breakdown of an AP site (a mixture of b-elimination, incubated overnight on LB plates at 37°C. The colonies were d-elimination and 3¢-OH products). Conversely, 25OXA was scored for surviving fractions. It is noted that the viability of stable in the heat/acid treatment (Fig. 2, lane 6) but was all the test strains decreased to ~10% after incubation with speci®cally cleaved by the ammonia/piperidine treatment acetate buffer (0.1 M, pH 4.6) alone. Thus, variations in (Fig. 2, lane 7). The products were 15mers bearing 3¢- viability depending on the strain were virtually negligible phosphate (d-elimination product) and 3¢-OH groups. 25G was in the incubation with acetate buffer alone. virtually intact in both treatments (Fig. 2, lanes 3 and 4). These results indicate that Xan and Oxa were site-speci®cally introduced into 25XAN and 25OXA, respectively, by the RESULTS DNA polymerase reaction. Site-speci®c introduction of Xan and Oxa into Repair activities of AlkA and Endo VIII for Xan and oligonucleotides Oxa Xan and Oxa were site-speci®cally incorporated into the The repair activities of various E.coli DNA repair enzymes for oligonucleotide substrates (25XAN and 25OXA, Table 1) by Xan and Oxa were examined using the nicking assay with the DNA polymerase reaction using dXTP and dOTP as 25XAN/COM25C and 25OXA/COM25C as substrates. The substrates. The N-glycosidic bond of Xan is fairly labile so enzymes tested included three bifunctional glycosylases that heat/acid treatment causes depurination, generating an AP containing N-glycosylase/AP lyase activities (Fpg, Endo III site. On the other hand, the N-glycosidic bond of Oxa is as and Endo VIII), two monofunctional N-glycosylases (AlkA stable as that of G to the heat/acid treatment. However, Oxa and Ung) and two AP endonucleases (Endo IV and Exo III). contains an O-acylisourea structure in the six-membered ring Among the enzymes tested, AlkA and Endo VIII recognized (1,3-oxazine ring) that undergoes a ring-opening reaction at Xan. The results of PAGE analysis of the reaction products of alkaline pH (pK = 9.4) (16). An extensive treatment of the AlkA and Endo VIII are shown in Figure 3. The treatment of ring-opened structure of Oxa induces decomposition of the 25XAN by AlkA (followed by Endo IV) resulted in a clear Oxa ring, though the ®nal decomposition product has not been band of a 3¢-OH product arising from cleavage at the identi®ed yet. Knowing the chemistry of Xan and Oxa, 25XAN and 25OXA prepared by the DNA polymerase introduced Xan site (Fig. 3A, lane 7). Similarly, the treatment reaction were treated by heat/acid or ammonia/piperidine as with Endo VIII resulted in a d-elimination product (Fig. 3B, Downloaded from https://academic.oup.com/nar/article-abstract/30/22/4975/2380497 by Ed 'DeepDyve' Gillespie user on 31 January 2018 Nucleic Acids Research, 2002, Vol. 30 No. 22 4979 Since Xan appeared to be a fair substrate of AlkA and Endo VIII, their kinetic parameters for Xan were determined from Michaelis±Menten plots and the results were compared with those for 7mG (AlkA) and Tg (Endo VIII). The K values of AlkA for 7mG and Xan were 31 and 53 nM, respectively (Table 2). The V values were 2.1 and 0.72 nM/min for 7mG max and Xan, respectively. These results indicated that AlkA had almost comparable af®nities (K ) for 7mG and Xan, but the catalytic rate for Xan was somewhat lower than for 7mG. The difference in the reaction ef®ciencies (V /K ) between 7mG max m and Xan was only 5-fold. Considering that 7mG is a physiological substrate of AlkA (30) and the relatively small difference in the reaction ef®ciencies, Xan is a fairly good substrate for AlkA. In contrast, Endo VIII showed a signi®- cantly lower reaction ef®ciency for Xan than for Tg, a physiological substrate of Endo VIII (31±33). The difference in the V /K values was ~50-fold. In terms of af®nity (K ), max m m Xan and Tg differed by 2.6-fold (124 versus 47 nM) and of catalytic rate (V ) by 19-fold (0.94 versus 18 nM/min). max Thus, Xan is a relatively poor substrate among those recognized by Endo VIII. N-glycosylase activity of AlkA and Endo VIII for Xan To con®rm the activity of AlkA and Endo VIII for Xan further, the N-glycosylase activity of both enzymes was examined by the release assay using 25XAN/COM25C containing [ H]Xan. After enzymatic treatment, the released product was separated from the substrate by gel ®ltration. Analysis of the radio- activity of the fraction from the gel ®ltration column showed that there was signi®cant release of the tritium count when Figure 3. Reaction products formed in the treatment of substrates contain- 25XAN/COM25C was incubated with AlkA and Endo VIII. ing Xan (25XAN) and Oxa (25OXA) by AlkA and Endo VIII. (A) AlkA However, the released tritium count was negligible when reactions. 100 fmol of 25G/COM25C, 25XAN/COM25C and 25OXA/ 3 3 control 25G/COM25C containing [ H]G instead of [ H]Xan COM25C (25G, 25XAN and 25OXA were 5¢- P labeled) were incubated was incubated with AlkA and Endo VIII. This was also the with 600 fmol of AlkA at 37°C for 30 min. After incubation, the sample was extracted with phenol and DNA was recovered by ethanol precipitation. case when 25XAN/COM25C was incubated with the reaction The sample was treated further with 120 fmol of Endo IV at 37°C for buffer alone. The pooled gel ®ltration fractions containing the 30 min. (B) Endo VIII reactions. 100 fmol of 25G/COM25C, 25XAN/ released product were further analyzed by reversed phase COM25C and 25OXA/COM25C were incubated with 600 fmol of Endo HPLC. In the HPLC analysis of the product released by AlkA VIII at 37°C for 30 min. In panels (A) and (B), the sample was separated by 16% denaturing PAGE. The substrates and enzymes used are indicated and Endo VIII, the major peak of the tritium count was eluted on the top of the gels. Lane 1 shows a 5¢- P-labeled marker (PRIM15). at 10.4 min (Fig. 4B and C) where authentic Xan (unlabeled) was eluted (Fig. 4A). Authentic G was eluted at 8.7 min under these conditions. Accordingly, AlkA and Endo VIII acted as lane 7). The observed incision products were consistent with N-glycosylases for 25XAN/COM25C, releasing a free base of the known catalytic modes of AlkA (+ Endo IV) and Endo VIII. Xan from the substrate. Although incision products of 25OXA by AlkA (+ Endo IV) Repair capacity of E.coli cell extracts for Xan and Endo VIII were barely detected with the same amount (600 fmol) of the enzymes as used for 25XAN (Fig. 3A and B, The repair capacity of the E.coli cell extracts for Xan was lane 5), treatment with an excessive amount of AlkA and Endo measured by the release assay. 25XAN/COM25C containing VIII (both 3 pmol) revealed their activity for Oxa (see Paired [ H]Xan was incubated with the cell extracts from E.coli base effects on the repair activity for Xan and Oxa). MV1161 (wild type) and MV1571 (alkA), and the released Table 2. Kinetic parameters of AlkA and Endo VIII for Xan b ±1 Enzyme Substrate K (nM) V (nM/min) V /K (min ) m max max m ±2 AlkA Xanthine 53 6 24 0.72 6 0.04 1.4 3 10 (1) ±2 7-Methylguanine 31 6 2 2.1 6 0.4 6.8 3 10 (4.9) ±3 Endo VIII Xanthine 124 6 33 0.96 6 0.26 7.6 3 10 (1) ±1 Thymine glycol 47 6 10 18 6 5 3.8 3 10 (50) The values are derived from three independent experiments and standard deviations are also indicated. The values in parentheses indicate relative values of V /K . max m Downloaded from https://academic.oup.com/nar/article-abstract/30/22/4975/2380497 by Ed 'DeepDyve' Gillespie user on 31 January 2018 4980 Nucleic Acids Research, 2002, Vol. 30 No. 22 Figure 5. Xan-releasing activity of the extracts from wild type and alkA mutant E.coli cells. The cell extracts were prepared from E.coli MV1161 (wild type) and MV1571 (alkA) treated without and with MNNG, respec- tively, as described in Materials and Methods. 25XAN/COM25C containing [ H]Xan (2.25 pmol) was incubated with 5 mg of the cell extracts at 37°C for 30 min. After incubation, the sample was loaded on to a Sephadex G-25 column and eluted with water. The amount of [ H]Xan released was counted on a liquid scintillation counter. Open columns, cells without MNNG treat- ment; closed columns, cells with MNNG treatment. The data are averages of three independent experiments. Standard deviations are indicated with error bars. Figure 4. N-glycosylase activity assays of AlkA and Endo VIII for Xan. (A) HPLC separation of authentic guanine (G) and Xan. Analysis was per- (Fig. 6A, lane 3). The activities for both pairs were compar- formed as described in Materials and Methods. (B) HPLC analysis of [ H]Xan released by AlkA. 2.25 pmol of 25XAN/COM25C containing able. The Xan:G and Xan:T pairs were very poor substrates of [ H]Xan was incubated with 3 pmol of AlkA at 37°C for 30 min. The AlkA (Fig. 6A, lanes 7 and 9). The order of activity for Xan released H-labeled material was separated from DNA by a Sephadex G-25 was C = A >> G > T with respect to the paired base. Unlike column. The column fractions containing the released H-labeled material AlkA, Endo VIII recognized Xan:C and Xan:G pairs with were pooled and evaporated. The sample was resuspended in a small volume of water and was subjected to HPLC analysis. HPLC analysis was comparable ef®ciency (Fig. 6B, lanes 7 and 10). The Xan:A performed as described in panel (A). (C) HPLC analysis of [ H]Xan and Xan:T pairs were very poor substrates. The order of released by Endo VIII. The experiment was performed in a similar manner activity was C = G >> A > T with respect to the paired base. using 6 pmol of Endo VIII. Compared to the reaction with Xan, a large excess of the enzymes (5- or 10-fold) and a long incubation time (1 h) were required to detect the activity for Oxa. For Oxa, AlkA and tritium count was measured after separation on a gel ®ltration Endo VIII exhibited a similar preference of the paired bases. column. However, no signi®cant difference in released The pairs of Oxa:A (Fig. 7, lanes 4 and 5), Oxa:C (Fig. 7, lanes radioactivity was observed between wild type and alkA 8 and 9) and Oxa:G (lanes 12 and 13) were comparable mutant cells (Fig. 5). It is possible that the basal level of substrates for AlkA and Endo VIII, whereas an Oxa:T pair was AlkA protein in the wild type cell was so low that the activity recognized poorly by the enzymes (Fig. 7, lanes 16 and 17). difference between the wild type and alkA mutant cells could Therefore, the order of the activity of both enzymes was be within an experimental ¯uctuation. The alkA gene is C = G = A > T with respect to the paired base. inducible by alkylating agents (34). Therefore, MV1161 and MV1571 cells were treated with MNNG, and the cell extracts Nitrous acid sensitivities of E.coli strains de®cient in from the two strains were subjected to the release assay. After AlkA and Endo VIII MNNG treatment, the wild type cell extract showed a 4-fold increase in Xan-releasing activity in comparison with the cell To assess the role of AlkA and Endo VIII in the repair of Xan extract without the MNNG treatment (Fig. 5). In contrast, such and Oxa in E.coli cells, the sensitivity of E.coli strains induction was not observed with the alkA mutant treated with MV1161 (wild type), MV1571 (alkA), KY100 (nei) and MNNG (Fig. 5). The results obtained with MNNG-treated KY101 (alkA nei) to nitrous acid was determined by treating cells were consistent with the data obtained with puri®ed them with sodium nitrite (NaNO ) pH 4.6 (29) under selective AlkA. conditions (i.e. in the presence of appropriate antibiotics). The single mutants defective in either the alkA or nei gene showed Paired base effects on the repair activity for Xan and nitrous acid sensitivities similar to those of the wild type cell. Oxa On the other hand, the alkA nei double mutant exhibited increased sensitivity (Fig. 8), implying a degenerative role of Xan and Oxa are potentially mutagenic lesions and pair with C AlkA and Endo VIII in counteracting the cytotoxic effect of and T during DNA replication (16±18). Thus, the ability of nitrous acid. In the above NaNO treatment, E.coli strains AlkA and Endo VIII to recognize Xan and Oxa paired with MV1161, MV1571 and KY100 were grown in the absence or different bases was examined. The activity was measured by the nicking assay using 25XAN/COM25N and 25OXA/ presence of one antibiotic (ampicillin or kanamycin), while COM25N (N = A, G, C, T) as substrates. AlkA recognized strain KY101 was grown in the presence of two antibiotics not only a Xan:C pair (Fig. 6A, lane 5) but also a Xan:A pair (ampicillin and kanamycin). The differences in growth Downloaded from https://academic.oup.com/nar/article-abstract/30/22/4975/2380497 by Ed 'DeepDyve' Gillespie user on 31 January 2018 Nucleic Acids Research, 2002, Vol. 30 No. 22 4981 Figure 7. Activities of AlkA and Endo VIII for Oxa paired with different bases. 25OXA/COM25N (N = A, G, C, T) (100 fmol) was incubated with 3 pmol of AlkA or Endo VIII for 1 h and products were analyzed as described in Figure 6. The substrates (base pairs) and enzymes used were indicated on the top of the gels. Lane 1 shows a 5¢- P-labeled marker (PRIM15). Figure 6. Activities of AlkA and Endo VIII for Xan paired with different Figure 8. Sensitivity of E.coli strains to nitrous acid. Escherichia coli cells bases. (A) AlkA reactions. 25XAN/COM25N (N = A, G, C, T) (100 fmol) pro®cient and de®cient in AlkA (encoded by the alkA gene) or Endo VIII was incubated with 300 fmol of AlkA at 37°C for 20 min followed by (encoded by the nei gene) were treated with the indicated concentrations of 120 fmol of Endo IV at 37°C for 30 min. (B) Endo VIII reactions. 25XAN/ NaNO in acetate buffer (pH 4.6) and the survival fractions were deter- COM25N (N = A, G, C, T) (100 fmol) was incubated with 600 fmol of 2 mined as described in Materials and Methods. Open circles, MV1161 (wild Endo VIII at 37°C for 1 h or 120 fmol of Endo IV at 37°C for 30 min. In type); closed circles, MV1571 (alkA); open triangles, KY100 (nei); closed (A) and (B), products were analyzed by 16% denaturing PAGE. The triangles, KY101 (alkA nei). The data are averages of three independent substrates (base pairs) and enzymes used are indicated on the top of the experiments. Standard deviations are indicated with error bars. gels. Lane 1 shows a 5¢- P-labeled marker (PRIM15). The 3¢-OH and d-elimination products are indicated by arrows. conditions (i.e. selective pressure) might have created an oxides such as NO and nitrous acid. For this purpose, we advantage for the strains MV1161, MV1571 and KY100 over prepared de®ned oligonucleotide substrates containing Xan the strain KY101 with respect to survival. Thus, NaNO and Oxa by DNA polymerase reactions with dXTP and dOTP. treatment was also performed in the absence of antibiotics by It has been reported that dXTP is a poor substrate of DNA taking advantage of the fact that the chromosomal markers for polymerase I (35). Our previous study has also shown that antibiotic resistance (ampicillin and kanamycin in this study) dOTP as well as dXTP are poorly utilized by DNA polymerase I are rarely lost even in the absence of selection pressure. relative to dGTP (19). However, the low but signi®cant However, the results obtained without antibiotics were usability of dXTP and dOTP by DNA polymerase I was good essentially similar to those obtained with antibiotics, hence enough for targeted incorporation of the nucleotides under ruling out the effects of selection pressure on survival appropriate DNA polymerase reaction conditions (Fig. 2). measurements. Among the seven enzymes tested, AlkA and Endo VIII recognized Xan and Oxa, though their activity for Oxa was lower than for Xan. Analysis of enzymatic parameters DISCUSSION revealed that the activity (V /K ) of AlkA for Xan is one- max m In the present study, we have screened the repair activity of ®fth of that for 7mG, a physiological substrate of AlkA E.coli enzymes for Xan and Oxa formed from G by nitrogen (Table 2). AlkA recognizes a variety of alkylated purine and Downloaded from https://academic.oup.com/nar/article-abstract/30/22/4975/2380497 by Ed 'DeepDyve' Gillespie user on 31 January 2018 4982 Nucleic Acids Research, 2002, Vol. 30 No. 22 pyrimidine lesions (34). It has been shown that 5-formyluracil complexed covalently to DNA have recently been solved and fragmented thymine residues are also substrates of AlkA (44,45). Although the overall structures of Endo VIII and Fpg (24,36±38). Thus, AlkA accepts structurally diverse base resemble each other, there are three dissimilar patches where lesions as substrates. Common features of the substrates amino acids are not conserved between the two enzymes. One recognized by AlkA are weak N-glycosidic bonds and explicit of them coincides with putative binding pockets for the or implicit positive charges induced in the base (24). A everted base. Thus, interactions in the dissimilar binding plausible catalytic mechanism of AlkA has been proposed pocket are likely to account for the distinct damage speci®city based on these features and the three-dimensional structure of of Endo VIII and Fpg. AlkA (39±41). In view of the weak N-glycosidic bond of Xan The activities of AlkA and Endo VIII for Xan and Oxa and the capacity of the active site of AlkA, it seems reasonable showed dependence on the base opposite the lesions, though that AlkA recognizes Xan as a substrate. Oxa was also a the activity for Oxa was consistently much lower than for Xan. substrate of AlkA but recognized poorly. The relative For Xan, the activity decreased in the following order of the hydrolysis rate of the N-glycosidic bond of Oxa is 44-fold paired base: C = A >> G > T for AlkA and C = G >> A > T for lower than that of Xan (16). Therefore, it is very likely that Endo VIII (Fig. 6). For Oxa, the order was C = G = A > T for such a difference affected the hydrolysis of the N-glycosidic both AlkA and Endo VIII (Fig. 7). Considering the base bond by AlkA and slowed down the reaction of Oxa relative pairing capacity of Xan and Oxa, C and T are the most to Xan. biologically relevant paired bases. The Xan:C and Oxa:C pairs The cell extracts from the E.coli strains pro®cient and can be formed by the reaction of a G:C pair with NO and de®cient in the alkA gene showed no signi®cant difference in nitrous acid, and Xan:T and Oxa:T pairs by misincorporation Xan-releasing activity. Possibly, the basal level of AlkA of T opposite these lesions during DNA replication (16±18). protein present in wild type cells was too low to exhibit Excision of Xan and Oxa from Xan:C and Oxa:C pairs, detectable activity over the experimental ¯uctuation in the respectively, by AlkA and Endo VIII and subsequent repair present assay. Although the attempt to show the difference in synthesis leads to restoration of genetic information, thus error Xan-releasing activity between wild type and alkA mutant free. Conversely, excision of Xan and Oxa from Xan:T and cells was unsuccessful, the repair activity for Xan in the wild Oxa:T pairs, respectively, results in mutation ®xation and type cell was clearly increased when the cell was treated with induces G:C®A:T transitions if it occurs in cells. According MNNG (Fig. 5). Such an increase was not observed with the to the present results (Figs 6 and 7), AlkA and Endo VIII alkA mutant when it was treated in the same manner, recognized Xan:C and Oxa:C pairs relatively well but Xan:T indicating that the induction of the repair activity for Xan in and Oxa:T pairs were poor substrates for the enzymes. Thus, cells was dependent on the alkA gene. These results suggest the poor activity of AlkA and Endo VIII for Xan:T and Oxa:T that AlkA can at least participate in the repair of Xan when it is pairs seems to be consistent with avoidance of mutation present at the induced level. The alkA gene is under the control ®xation in cells. This is in contrast to the preference of E.coli of the ada gene and inducible by the alkylating agent such as Endo V, which recognizes all four Xan:N pairs (N = A, G, C, MNNG (34). However, no information is currently available T) with a preference for the Xan:T pair (21). Endo V was on whether NO or nitrous acid induces the alkA gene through originally found as a deoxyinosine endonuclease that incises this pathway. the second phosphodiester bond on the 3¢ side of deoxyinosine In this study, it has also been shown that Endo VIII has (a deoxyribonucleoside form of Hx) (7). More recently it has repair activity for Xan. The activity of Endo VIII for Xan been shown to have a broad substrate speci®city including Hx, measured by enzymatic parameters was low relative to that for U, Xan, AP site and mismatched bases (7,21,46,47). its physiological substrate (Tg) (Table 2). However, the low In the present work, it has been shown that the alkA nei but signi®cant activity may be crucial for repair of Xan when double mutant, but not the alkA and nei single mutants, was the level of AlkA is not suf®ciently high, e.g. in uninduced sensitive to nitrous acid (Fig. 8). These results suggest a cells. Endo VIII was originally found as a repair enzyme for degenerative role of AlkA and Endo VIII in the repair of a Tg and urea residues (31), and later it was shown to recognize certain class of nitrous acid-induced DNA lesions, for example other oxidative pyrimidine damage such as 5-hydroxycyto- Xan and Oxa, though involvement of other E.coli base sine, 5-hydroxyuracil and uracil glycol (33,42). Thus, sub- excision repair enzymes such as MutY, Mug and TagI has not strate speci®city of Endo VIII is similar to that of Endo III. been eliminated in this study. The actual mechanisms However, the amino acid sequence of Endo VIII shows no operating in E.coli cells to counteract the lethal and mutagenic homology to that of Endo III. Rather, the N-terminal and effects of nitrous acid and NO appear to be rather complicated. C-terminal regions of Endo VIII are similar to those of Fpg Several studies show cell killing and mutagenicity by nitrous involved in repair of oxidative purine lesions in E.coli (32). acid or NO increase in the cells de®cient in nucleotide Furthermore, Endo VIII has been shown recently to excise excision repair (48±50) or recombination repair (51). In oxidative purine lesions including 7,8-dihydro-8-oxoguanine contrast, studies on n® (the Endo V gene) mutants by Weiss's and formamidopyrimidine (25,43). Thus, Endo VIII has a group indicate the involvement of Endo V in avoidance of potential capacity to accept purine lesions as well as nitrous acid-induced mutations (52±54). Thus, at least four pyrimidine lesions, though the activities may vary. In this repair pathways including base excision repair indicated by context, it may not be surprising that Endo VIII recognized this study, nucleotide excision repair, recombination repair, Xan and Oxa. However, questions remain as to why Xan was a and repair initiated by Endo V have been implicated in better substrate than Oxa for Endo VIII and why Xan and Oxa restoration of DNA lesions formed by nitrous acid and NO. It were substrates for Endo VIII but not for Fpg. Concerning the is not clear whether such differences originate from dosage or latter, the three-dimensional structures of Endo VIII and Fpg types of nitrogen oxide species, cell conditions or accessed Downloaded from https://academic.oup.com/nar/article-abstract/30/22/4975/2380497 by Ed 'DeepDyve' Gillespie user on 31 January 2018 Nucleic Acids Research, 2002, Vol. 30 No. 22 4983 19. Suzuki,T., Yoshida,M., Yamada,M., Ide,H., Kobayashi,M., Kanaori,K., phenotypes (lethal or mutation effect). Thus, further studies Tajima,K. and Makino,K. (1998) Misincorporation of 2¢-deoxyoxanosine are necessary to clarify the whole picture of the cellular 5¢-triphosphate by DNA polymerases and its implication for mutagenesis. mechanisms to counteract mutagenic and lethal effects Biochemistry, 37, 11592±11598. associated with nitrogen oxide species. 20. Suzuki,T., Yamada,M., Ide,H., Kanaori,K., Tajima,K., Morii,T. and Makino,K. (2000) Identi®cation and characterization of a reaction product of 2¢-deoxyoxanosine with glycine. Chem. Res. Toxicol., 13, ACKNOWLEDGEMENTS 227±230. 21. He,B., Qing,H. and Kow,Y.W. (2000) Deoxyxanthosine in DNA is This research was supported by Grants-in-Aid from the repaired by Escherichia coli endonuclease V. Mutat. Res., 459, 109±144. Ministry of Education, Culture, Sports, Science and 22. Wang,G., Palejwala,V.A., Dunman,P.M., Aviv,D.H., Murphy,H.S., Rahman,M.S. and Humayun,M.Z. (1995) Alkylating agents induce Technology, Japan (to H.I. and K.M.). UVM, a recA-independent inducible mutagenic phenomenon in Escherichia coli. Genetics, 141, 813±823. 23. Saito,Y., Uraki,F., Nakajima,S., Asaeda,A., Ono,K., Kubo,K. and REFERENCES Yamamoto,K. (1997) Characterization of endonuclease III (nth) and 1. Frederico,L.A., Kunkel,T.A. and Shaw,B.R. (1990) A sensitive genetic endonuclease VIII (nei) mutants of Escherichia coli K-12. J. Bacteriol., assay for the detection of cytosine deamination: determination of rate 179, 3783±3785. constants and the activation energy. Biochemistry, 29, 2532±2537. 24. Masaoka,A., Terato,H., Kobayashi,M., Honsho,A., Ohyama,Y. and 2. Lindahl,T. (1993) Instability and decay of the primary structure of DNA. Ide,H. (1999) Enzymatic repair of 5-formyluracil. I. Excision of 5- Nature, 362, 709±715. formyluracil site-speci®cally incorporated into oligonucleotide substrates 3. Duncan,B.K. and Weiss,B. (1982) Speci®c mutator effects of ung (uracil- by AlkA protein (Escherichia coli 3-methyladenine DNA glycosylase II). DNA glycosylase) mutations in Escherichia coli. J. Bacteriol., 151, J. Biol. Chem., 274, 25136±25143. 750±755. 25. Asagoshi,K., Yamada,T., Okada,Y., Terato,H., Ohyama,Y., Seki,S. and 4. Hill-Perkins,M., Jones,M.D. and Karran,P. (1986) Site-speci®c Ide,H. (2000) Recognition of formamidopyrimidine by Escherichia coli mutagenesis in vivo by single methylated or deaminated purine bases. and mammalian thymine glycol glycosylases. J. Biol. Chem., 275, Mutat. Res., 162, 153±163. 24781±24786. 5. Friedberg,E.C., Walker,G.C. and Siede,W. (1995) DNA Repair and 26. Sarker,A.H., Ikeda,S., Nakano,H., Terato,H., Ide,H., Imai,K., Mutagenesis. ASM Press, Washington, DC. Akiyama,K., Tsutsui,K., Bo,Z., Kubo,K., Yamamoto,K., Yasui,A., 6. Saparbaev,M. and Laval,J. (1994) Excision of hypoxanthine from DNA Yoshida,M.C. and Seki,S. (1998) Cloning and characterization of a containing dIMP residues by the Escherichia coli, yeast, rat and human mouse homologue (mNthl1) of Escherichia coli endonuclease III. alkylpurine DNA glycosylases. Proc. Natl Acad. Sci. USA, 91, J. Mol. Biol., 282, 761±774. 5873±5877. 27. Asagoshi,K., Odawara,H., Nakano,H., Miyano,T., Terato,H., Ohyama,Y., 7. Yao,M., Hatahet,Z., Melamede,R.J. and Kow,Y.W. (1994) Puri®cation Seki,S. and Ide,H. (2000) Comparison of substrate speci®cities of and characterization of a novel deoxyinosine-speci®c enzyme, Escherichia coli endonuclease III and its mouse homologue (mNTH1) deoxyinosine 3¢ endonuclease, from Escherichia coli. J. Biol. Chem., using de®ned oligonucleotide substrates. Biochemistry, 39, 269, 16260±16268. 11389±11398. 8. Lindahl,T. (1979) DNA glycosylases, endonuclease for apurinic/ 28. Ide,H., Okagami,M., Murayama,H., Kimura,Y. and Makino,K. (1993) apyrimidinic sites and base excision repair. Prog. Nucleic Acid Res. Mol. Synthesis and characterization of oligonucleotides containing the alpha- Biol., 22, 135±192. anomer of deoxyadenosine to study its in¯uence on DNA replication. 9. Marletta,M.A. (1989) Nitric oxide: biosynthesis and biological Biochem. Mol. Biol. Int., 31, 485±491. signi®cance. Trends Biochem. Sci., 14, 488±492. 29. Miller,J.H. (1972) Experiments in Molecular Genetics. Cold Spring 10. Ohshima,H. and Bartsch,H. (1994) Chronic infections and in¯ammatory Harbor Laboratory Press, Cold Spring Harbor, NY. processes as cancer risk factors: possible role of nitric oxide in 30. Thomas,L., Yang,C.-H. and Goldthwait,D.A. (1982) Two DNA carcinogenesis. Mutat. Res., 305, 253±264. glycosylases in Escherichia coli which release primarily 3- 11. Wink,D.A., Vodovots,Y., Laval,J., Laval,F., Dewhirst,M.W. and methyladenine. Biochemistry, 21, 1162±1169. Mitchell,J.B. (1998) The multifaceted roles of nitric oxide in cancer. 31. Melamede,R.J., Hatahet,Z., Kow,Y.W., Ide,H. and Wallace,S.S. (1994) Carcinogenesis, 19, 711±721. Isolation and characterization of endonuclease VIII from Escherichia 12. Wink,D.A., Kasprzak,K.S., Maragos,C.M., Elespuru,R.K., Misra,M., coli. Biochemistry, 33, 1255±1264. Dunams,T.M., Cebula,T.A., Koch,W.H., Andrews,A.W., Allen,J.S. and 32. Jiang,D., Hatahet,Z., Blaisdell,J.O., Melamede,R.J. and Wallace,S.S. Keefer,L.K. (1991) DNA deaminating ability and genotoxicity of nitric (1997) Escherichia coli endonuclease VIII: cloning, sequencing, and oxide and its progenitors. Science, 254, 1001±1003. overexpression of the nei structural gene and characterization of nei and 13. Suzuki,T., Yamaoka,R., Nishi,M., Ide,H. and Makino,K. (1996) Isolation nei nth mutants. J. Bacteriol., 179, 3773±3782. and characterization of a novel product, 2¢-deoxyoxanosine, from 2¢- 33. Jiang,D., Hatahet,Z., Melamede,R.J., Kow,Y.W. and Wallace S.S. (1997) deoxyguanosine, oligodeoxynucleotide, and calf thymus DNA treated by Characterization of Escherichia coli endonuclease VIII. J. Biol. Chem., nitrous acid and nitric oxide. J. Am. Chem. Soc., 118, 2515±2516. 272, 32230±32239. 14. Lucas,L.T., Gatehouse,D. and Shuker,D.E. (1999) Ef®cient nitroso group 34. Lindahl,T., Sedgwick,B., Sekiguchi,M. and Nakabeppu,Y. (1988) transfer from N-nitrosoindoles to nucleotides and 2¢-deoxyguanosine at Regulation and expression of the adaptive response to alkylating agents. physiological pH. J. Biol. Chem., 274, 18319±18326. Annu. Rev. Biochem., 57, 133±157. 15. Suzuki,T., Ide,H., Yamada,M., Endo,N., Kanaori,K., Tajima,K., Morii,T. 35. Bessman,M.J., Lehman,I.R., Adler,J., Zimmerman,S.B., Simms,E.S. and and Makino,K. (2000) Formation of 2¢-deoxyoxanosine from 2¢- Kornberg,A. (1958) Enzymatic synthesis of deoxyribonucleic acid. III. deoxyguanosine and nitrous acid: mechanism and intermediates. Nucleic The incorporation of pyrimidine and purine analogues into Acids Res., 28, 544±551. deoxyribonucleic acid. Proc. Natl Acad. Sci. USA., 44, 633±640. 16. Suzuki,T., Matsumura,Y., Ide,H., Kanaori,K. Tajima,K. and Makino,K. 36. Bjelland,S., Birkeland,N.K., Benneche,T., Volden,G. and Seeberg,E. (1997) Deglycosylation susceptibility and base-pairing stability of 2¢- (1994) DNA glycosylase activities for thymine residues oxidized in the deoxyoxanosine in oligonucleotide. Biochemistry, 36, 8013±8019. methyl group are functions of the AlkA enzyme in Escherichia coli. 17. Eritja,R. Horowitz,D.M., Walker,P.A., Ziehler-Martin,J.P., J. Biol. Chem., 269, 30489±30495. Boosalis,M.S., Goodman,M.F., Itakura,K. and Kaplan,B.E. (1986) 37. Terato,H., Masaoka,A., Kobayashi,M., Fukushima,S., Ohyama,Y., Synthesis and properties of oligonucleotides containing 2¢- Yoshida,M. and Ide,H. (1999) Enzymatic repair of 5-formyluracil II. deoxynebularine and 2¢-deoxyxanthosine. Nucleic Acids Res., 24, Mismatch formation between 5-formyluracil and guanine during DNA 168±175. replication and its recognition by two proteins involved in base excision 18. Kamiya,H., Shimizu,M. Inoue,H. and Ohtsuka,E. (1992) Mutation induced by deoxyxanthosine in codon 12 of a synthetic c-Ha-ras gene. repair (AlkA) and mismatch repair (MutS). J. Biol. Chem., 274, Nucl. Nucl., 11, 247±260. 25144±25150. Downloaded from https://academic.oup.com/nar/article-abstract/30/22/4975/2380497 by Ed 'DeepDyve' Gillespie user on 31 January 2018 4984 Nucleic Acids Research, 2002, Vol. 30 No. 22 38. Privezentzev,C.V., Saparbaev,M., Sambandam,A., Greenberg,M.M. and 46. Yao,M. and Kow,Y.W. (1995) Interaction of deoxyinosine 3¢- Laval,J. (2000) AlkA protein is the third Escherichia coli DNA repair endonuclease from Escherichia coli with DNA containing deoxyinosine. protein excising a ring fragmentation product of thymine. Biochemistry, J. Biol. Chem., 270, 28609±28616. 39, 14263±14268. 47. Yao,M. and Kow,Y.W. (1997) Further characterization of Escherichia 39. Yamagata,Y., Kato,M., Odawara,K., Tokuno,Y., Nakashima,Y., coli endonuclease V. J. Biol. Chem., 272, 30774±30779. Tatsushima,N., Yasumura,K., Tomita,K., Ihara,K., Fujii,Y., 48. Hartman,Z., Henrickson,E.N., Hartman,P.E. and Cebula,T.A. (1994) Nakabeppu,Y., Sekiguchi,M. and Fujii,S. (1996) Three dimensional Molecular models that may account for nitrous acid mutagenesis in structure of a DNA repair enzyme, 3-methyladenine DNA glycosylase II, organisms containing double-stranded DNA. Environ. Mol. Mutagen., from Escherichia coli. Cell, 86, 311±319. 24, 168±175. 40. Labahn,J., Scharer,O.D., Long,A., Ezaz-Nikpay,K., Verdine,G.L. and 49. Sidorkina,O., Saparbaev,M. and Laval,J. (1997) Effects of nitrous acid Ellenberger,T.E. (1996) Structure basis for the excision repair of treatment on the survival and mutagenesis of Escherichia coli cells alkylation-damaged DNA. Cell, 86, 321±329. lacking base excision repair (hypoxanthine-DNA glycosylase-ALK A 41. Hollis,T., Ichikawa,Y. and Ellenberger,T. (2000) DNA bending and a protein) and/or nucleotide excision repair. Mutagenesis, 12, 23±28. ¯ip-out mechanism for base excision by the helix±hairpin±helix DNA 50. Tamir,S., Burney,S. and Tannenbaum,S.R. (1996) DNA damage by nitric glycosylase, Escherichia coli AlkA. EMBO J., 19, 758±766. oxide. Chem. Res. Toxicol., 9, 821±827. 42. Purmal,A.A., Lampman,G.W., Bond,J.P., Hatahet,Z. and Wallace S.S. 51. Spek,E.J., Wright,T.L., Stitt,M.S., Taghizadeh,N.R., Tannenbaum,S.R., (1998) Enzymatic processing of uracil glycol, a major oxidative product Marinus,M.G. and Engelward,B.P. (2001) Recombinational repair is of DNA cytosine. J. Biol. Chem., 273, 10026±10035. critical for survival of Escherichia coli exposed to nitric oxide. 43. Blaisdell,J.O., Hatahet,Z. and Wallace,S.S. (1999) A novel role for J. Bacteriol., 183, 131±138. Escherichia coli endonuclease VIII in prevention of spontaneous G®T 52. Guo,G. and Weiss,B. (1998) Endonuclease V (n®) mutant of Escherichia transversions. J. Bacteriol., 181, 6396±6402. coli K-12. J. Bacteriol., 180, 46±51. 44. Zharkov,D.O., Golan,G., Gilboa,R., Fernandes,A.S., Gerchman,S.E., 53. Schouten,K.A. and Weiss,B. (1999) Endonuclease V protects Kycia,J.H., Rieger,R.A., Grollman,A.P. and Shoham,G. (2002) Structural Escherichia coli against speci®c mutations caused by nitrous acid. analysis of an Escherichia coli endonuclease VIII covalent reaction Mutat. Res., 435, 245±254. intermediate. EMBO J., 21, 789±800. 54. Weiss,B. (2001) Endonuclease V of Escherichia coli prevents mutations 45. Gilboa,R., Zharkov,D.O., Golan,G., Fernandes,A.S., Gerchman,S.E., from nitrosative deamination during nitrate/nitrite respiration. Matz,E., Kycia,J.H., Grollman,A.P. and Shoham,G. (2002) Structure of Mutat. Res., 461, 301±309. formamidopyrimidine-DNA glycosylase covalently complexed to DNA. J. Biol. Chem., 277, 19811±19816. Downloaded from https://academic.oup.com/nar/article-abstract/30/22/4975/2380497 by Ed 'DeepDyve' Gillespie user on 31 January 2018

Journal

Nucleic Acids ResearchOxford University Press

Published: Nov 15, 2002

There are no references for this article.