Oxidative base damage to DNA: specificity of base excision repair enzymes

Mutation Research 462 Ž2000. 121–128 www.elsevier.comrlocaterreviewsmr Community address: www.elsevier.comrlocatermutres

Oxidative base damage to DNA: specificity of base excision repair enzymes Jean Cadet ) , Anne-Gaelle D’Ham, Victor Duarte, Didier Gasparutto, ¨ Bourdat, Cedric ´ Anthony Romieu, Jean-Luc Ravanat Departement de Recherche Fondamentale sur la Matiere des Acides Nucleiques’’, ´ ` Condensee, ´ SCIBr Laboratoire ‘‘Lesions ´ ´ CEA r Grenoble, 17 AÕenue des Martyrs, Grenoble Cedex 9 F-38054, France Received 2 July 1999; received in revised form 17 August 1999; accepted 17 August 1999

Abstract Base excision repair ŽBER. is likely to be the main mechanism involved in the enzymatic restoration of oxidative base lesions within the DNA of both prokaryotic and eukaryotic cells. Emphasis was placed in early studies on the determination of the ability of several bacterial DNA N-glycosylases, including Escherichia coli endonuclease III Žendo III. and formamidopyrimidine DNA N-glycosylase ŽFpg., to recognize and excise several oxidized pyrimidine and purine bases. More recently, the availability of related DNA repair enzymes from yeast and human has provided new insights into the enzymatic removal of several POH-mediated modified DNA bases. However, it should be noted that most of the earlier studies have involved globally modified DNA as the substrates. This explains, at least partly, why there is a paucity of accurate kinetic data on the excision rate of most of the modified bases. Interestingly, several oxidized pyrimidine and purine nucleosides have been recently inserted into defined sequence oligonucleotides. The use of the latter substrates, together with overexpressed DNA N-glycosylases, allows detailed studies on the efficiency of the enzymatic release of the modified bases. This was facilitated by the development of accurate chromatographic and mass spectrometric methods aimed at measuring oxidized bases and nucleosides. As one of the main conclusions, it appears that the specificity of both endo III and Fpg proteins is much broader than expected a few years ago. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Oxidized bases; DNA N-glycosylases; Endonucleases; Modified oligonucleotides

X AbbreÕiations: dAdo, 2 -deoxyadenosine; DHdThd, 5,6-dihydrothymidine; DHThy, 5,6-dihydrothymine; dF, N-Ž2-deoxy-b-D–erythroX . pentofuranosyl formamide; dGuo, 2 -deoxyguanosine; dZ, 2,2-diamino-4-wŽ2-deoxy-b-D-erythro-pentofuranosyl.aminox-5Ž2 H .-oxazolone; X FapyGua, 2,6-diamino-4-hydroxy-5-formamidopyrimidine; 5-OHdCyd, 5-hydroxy-2 -deoxycytidine; 5-OHCyt, 5-hydroxycytosine; 5-OHUra, X X 5-hydroxyuracil; 5-OHdUrd, 5-hydroxy-2 -deoxyuridine; 8-oxodGuo, 8-oxo-7,8-dihydro-2 -deoxyguanosine; 8-oxoGua, 8-oxo-7,8-dihydroguanine; Tg, 5,6-dihydroxy-5,6-dihydrothymine; Ug, 5,6-dihydroxy-5,6-dihydroxyuracil; BER, base excision repair; ECD, electrochemical detection; endo III, endonuclease III; ESR, electron spin resonance; Fpg, formamidopyrimidine DNA N-glycosylase; GC-MS, gas chromatography mass spectrometry; HPLC, high-performance liquid chromatography; MALDI-TOF MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; NER, nucleotide excision repair; PAGE, polyacrylamide gel electrophoresis ) Corresponding author. Tel.: q33-4-76-88-49-87; fax: q33-4-76-88-50-90; e-mail: [email protected]

1383-5742r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 3 - 5 7 4 2 Ž 0 0 . 0 0 0 2 2 - 3

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1. Introduction DNA is the major cellular target of one-electron oxidation process and various reactive oxygen and nitrogen species including POH radical, singlet oxygen and peroxynitrite, at least, in terms of mutagenesis and carcinogenesis w1x. Modified purine and pyrimidine bases constitute one of the major classes of oxidative DNA damage together with oligonucleotide strand breaks, DNA–protein cross-links and abasic sites. During the last two decades, major efforts were devoted to the isolation and characterization of the main stable oxidation products of DNA bases using isolated DNA and related model compounds w1x. Additional insights in the mechanistic features of the formation of the latter DNA modifications were gained from electron spin resonance ŽESR. and time-resolved spectroscopic studies on the transient radicals involved in most of these oxidation reactions w2,3x. Interestingly, up to 40 modified bases including diastereoisomers have been identified in model studies wfor comprehensive reviews, see Refs. w1,4xx. Unfortunately, only a few base lesions including 8-oxo-7,8-dihydroguanine Ž8-oxoGua., 2,6-diamino-4-hydroxy-5-formamidopyrimidine ŽFapyGua. and 5-hydroxycytosine Ž5-OHCyt. have been measured accurately within cellular DNA due to the lack, until recently, of accurate and sensitive assays w4,5x. Another important field of research which is still receiving a great deal of attention concerns the repair of oxidized DNA bases. Elimination of the bulk of the latter class of lesions appears to mainly proceed through the base excision repair ŽBER. pathway. This involves the cleavage of the N-glycosidic bond of the oxidized bases by various enzymes and subsequent removal of the resulting abasic site by endonuclease activities. Several DNA N-glycosylases, which were claimed initially to have non-overlapping substrate specificities, have been isolated and characterized w6–8x. These mostly include prokaryotic, endonuclease III Žendo III. w9,10x, endonuclease VIII w11x and formamidopyrimidine DNA N-glycosylase ŽFpg. w12x. It was also shown that 3-methyladenine DNA glycosylase II ŽAlkA. is able to excise 5-formyluracil and, to a lesser extent, 5-Žhydroxymethyl.uracil w13x, two oxidation compounds of the methyl group of thymine. On the other hand, implication of the nucleotide excision repair ŽNER. path-

way, which is efficient for the removal of bulky adducts, appears to be marginal for oxidized bases even if 5,6-dihydroxy-5,6-dihydrothymine ŽTg. has been shown to be a substrate of the bacterial UvrABC nuclease complex w14,15x. Interestingly, evidence was recently provided for the occurrence of transcription-coupled repair of oxidative base damage in yeast and human cells w16,17x. The present short survey will focus on studies aimed at assessing the substrate specificity of the two main classes of Escherichia coli enzymes, namely endo III and Fpg, which are involved in the BER pathways of oxidized bases and related radiation-induced degradation products. Emphasis is placed on accurate kinetic determinations, including the measurement of apparent K m and Vm associated with the glycosylase-mediated release of modified bases. Interestingly, E. coli was found, more than 20 years ago, to possess an endonuclease activity capable of nicking UV-irradiated DNA w18x, likely at sites of 6-hydroxy-5,6-dihydrocytosine w19x. The ability for Fpg to act as a N-glycosylase was first established on imidazole ring-opened purine bases including 2,6-diamino-4-hydroxy-5-Ž N-methyl.formamidopyrimidine, FapyGua and 4,6-diamino-5-formamidopyrimidine w20–22x. Qualitative information on the specificity of endo III and Fpg enzymes was gained for the bulk from studies involving naked DNA which was modified by various treatments including exposure to ionizing radiation and oxidizing agents w22–25x. Interestingly, the gene of both repair enzymes has been cloned, allowing the availability of high-purity proteins in relatively large amounts w26,27x.

2. Overall strategy for the determination of the substrate specificity of endo III and Fpg proteins As already mentioned, most of the studies aimed at delineating the specificity of DNA N-glycosylases for oxidized bases have been performed until recently using isolated DNA that contains several modifications. The availability of short DNA fragments that contain a unique modification in a defined sequence context allows accurate determination and comparison of kinetic parameters of excision. The early version of the gas chromatography mass spec-

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trometry ŽGC-MS. assay has been extensively used for the measurement of N-glycosylase-mediated base release from modified DNA w22–25,28–32x. However, the presence of salts in the enzymatic reaction, which are known to prevent efficient sylilation of the released modified bases, may affect the quantitative aspect of the analysis, particularly when internal standards are not used. An improved method Žvide infra., together with the promising high-performance liquid chromatography ŽHPLC.rMS-MS assay w33x, is now available for this purpose. 2.1. Preparation of site-specific modified oligonucleotides The preparation of defined sequence DNA fragments that contain one or two modified bases at specific sites has received increasing attention during the last decade. One major difficulty for the chemical insertion of oxidized nucleotides is the presence of additional functional groups which have to be protected during the solid phase synthesis of oligonucleotides. In addition, most of these compounds are more unstable than the parent molecules during the conditions of deprotection particularly those requiring alkali treatment. Therefore, modification and optimization of synthesis procedures have been designed to insert several oxidized nucleosides into oligonucleotides. Site-specifically incorporated nucleosides include 5-hydroxy-2X-deoxyuridine Ž5OHdUrd . w34,35 x, 5-hydroxy-2 X-deoxycytidine Ž5-OHdCyd. w35,36x, N-Ž2-deoxy-b-D-erythro-pentofuranosyl.formamide ŽdF. w37,38x, 8-oxo-7, 8dihydro-2X-deoxyadenosine w39x, 8-oxo-7,8-dihydro-2X-deoxyguanosine Ž8-oxodGuo. w40,41x and the 4 R and 4S diastereomers of 4-hydroxy-8-oxo-4,8-dihydro-2X-deoxyguanosine w42x. On the other hand, the insertion of 5,6-dihydrothymidine ŽDHThd. w43x and N- Ž 2-deoxy-b- D -erythro-pentofuranosyl . cyanuric acid w44x, which are quite stable, is almost straightforward. In addition, several oligonucleotides that contain a tandem damage, including 8-oxodGuodF, dF-8-oxodGuo w45x and the two 5X R and 5X S diastereomers of both 5X 8-cyclopurine derivatives of dAdo and dGuo w46,47x, were recently prepared by chemical synthesis. However, alkali labile compounds such as 5,6-dihydroxy-5,6-dihydrothymidine, 5,6-dihydroxy-5,6-dihydro-2X-deoxyuridine and 2,

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2-diamino-4-wŽ2-deoxy-b-D-erythro-pentofuranosyl.aminox-5Ž2 H .-oxazolone ŽdZ. could not be incorporated into DNA fragments using the solid phase synthetic approach. The thymidine- and 2X-deoxyuridine glycol-containing oligonucleotides were prepared by postsynthetic oxidation of a unique thymine w48x and uracil w49x residue within short oligonucleotides, respectively. Specific riboflavinmediated photosensitization of the unique guanine base of oligonucleotides was successfully applied to introduce a dZ lesion in 9- and 15-mers w50x. Interestingly Ž5R .-5-hydroxy-5,6-dihydrothymidine-containing oligonucleotides were prepared by UV photolysis of appropriate precursors which were initially incorporated into the DNA fragments w51x. Subsequent alkali treatment of the latter oligomers was found to generate in good yield N-Ž2-deoxy-b-Derythro-pentofuranosyl.-N-3-Ž2 R-hydroxyisobutyric acid.urea w52x. It should be added that enzymatic incorporation of the triphosphate derivatives of 5OHdCyd, 5-OHdUrd and DHThd has been used to prepare the related site-specifically modified oligonucleotides w53,54x. 2.2. Optimized assays for measuring the excision of the modified bases Excision of the modified bases as a result of the glycosylase activity of Fpg and endo III is accompanied by endonuclease-mediated cleavage of the related phosphodiester bond w6–8x. A b–d elimination mechanism is involved in the associated b-lyase activity of the Fpg protein leading to DNA strand cleavage. On the other hand, a b-elimination process has been suggested to explain the removal of the abasic sites generated by endo III with subsequent cleavage of the phosphodiester bond on the 3X side. The quantitation of DNA nicks and therefore the assessment of the glycosylase activity of both Fpg and endo III enzymes are usually achieved by applying polyacrylamide gel electrophoresis ŽPAGE. analysis under denaturating conditions w55,56x. The latter suitable approach, which requires the w32 Px labeling of the DNA probes, does not provide structural information on the released bases. This can be achieved by applying an accurate HPLCrGC-MS assay w57x, which also allows the measurement of the residual non-excised damage in the DNA fragment.

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Interestingly, using the latter method, we noticed a complete balance of 5,6-dihydrothymine ŽDHThy. released by endo III in the supernatant and still present in the DNA fragment w43x. Only small amounts of 5-OHCyt were measured in incubation mixtures of Fpg and endo III and 5-OHdCyd-containing oligonucleotide. In contrast, more efficient cleavage of the latter probe was observed as inferred from the PAGE analysis. This raises the question of the structure of the released modified base.

3. Endo III and Fpg-mediated excision of single base lesions from defined sequence duplex DNA fragments Determination of the substrate specificity of endo III and Fpg proteins has required the accurate measurement of the kinetic parameters of excision of various base lesions from defined sequence oligonucleotides that contain an unique damage. The sitespecifically modified substrates were annealed with the complementary strand with possible changes in the base opposite the lesion. Michaelis constants, including K m and Vm , were obtained using either the PAGE assay andror the HPLCrGC-MS and HPLCrECD Želectrochemical detection. methods. Thus, it was established that 8-oxoGua is a relevant substrate for Fpg w58x, whereas 8-oxo-7,8-dihydroadenine is, at best, barely excised by the latter enzyme w58,59x. More recently, the relative efficacy for endo III to remove three oxidized bases, including 5,6-dihydroxy-5,6-dihydrouracil ŽUg., 5-hydroxyuracil Ž5-OHUra. and 5-OHCyt, has been assessed from the comparison of the ratio between Vm and K m . The relative efficiency of excision is the following Ug Ž1.0. ) 5-OHCyt Ž0.74. ) 5-OHUra Ž0.53.. In another study, kinetic parameters of endo III- and Fpg-mediated excision were determined for 5-OHCyt, DHThy and Tg w43x. If 5-OHCyt is taken as the reference compound, the relative efficiency of excision by endo III decreases in the following order Tg Ž7.0. ) -OHCyt Ž1. ) HThy Ž0.63.. It was also found that the Fpg protein is able to excise the three latter base lesions with the following relative efficacy for DHThy Ž0.26. and 5-OHCyt Ž0.68. with respect to that of 8-oxoGua. Interestingly, N wŽ2 R .-

2-hydroxyisobutyric acidxurea, the opened-ring product of Ž5R .-5-hydroxy-5,6-dihydrothymine, was found to be a good substrate for Fpg since the efficiency of its excision was found to be about half of that of 8-oxoGua w60x. This also applies to the oxazolone lesion, which is a predominant POH and one-electron oxidation product of the guanine moiety of DNA w4,50,61,62x. The ratio Vm rK m for the excision of the latter compound was found to be only four times lower than that of 8-oxoGua. Interestingly it should be mentioned that both the oxazolone lesion and formamide, a major POH-mediated degradation product of thymine and cytosine, are good substrates for endo III. In contrast, cyanuric acid, which is a singlet oxygen oxidation product of 8-oxoGua, is not excised by either Fpg or endo III w44x. It should be mentioned that the first striking evidence of a broader substrate specificity for endonuclease III and Fpg proteins was provided by a repair study involving site-specifically incorporated 5-OHdCyd and 5OHdUrd oligonucleotides. It was found that both enzymes were able to excise the oxidized cytosine lesions w63x.

4. Endo III and Fpg-mediated excision of double lesions from defined sequence duplex DNA Complex damage, including either two modified bases or the association of a base damage with a strand break or an abasic site, is expected to be generated by ionizing radiation within cellular DNA w1x. There are only a few available data on the ability of Fpg and endo III proteins to deal with such DNA lesions. Several duplex DNA fragments, which contain DHThy on one strand and an abasic site on the other strand with the two lesions being one, three, five and seven bases apart, have been constructed w54x. Interestingly, it was found that the N-glycosylase activity of endo III on DHThy was inhibited when the two lesions were located in the close vicinity. In contrast, the AP lyase activity was not affected by the presence of the base damage. Recently, dF and 8-oxodGuo have been incorporated as tandem base lesions into 25-mers with the two possible sequences, dFr8-oxodGuo and 8-oxodGuordF w45x. It was found that Fpg and endo III were able to

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specifically excise 8-oxoGua and formamide, respectively, from the former modified oligonucleotide. Incubation of 8-oxodGuordF duplex Ž25 bp. with Fpg led to partly different results since in addition to the removal of 8-oxoGua, excision of the two modified bases was observed.

5. Conclusion Increasing information on the substrate specificity of both Fpg and endo III has been recently gained from various studies involving the use of site-specifically modified oligonucleotides. Interestingly, kinetic parameters are available for various modified purine and pyrimidine bases. One first major observation is that both repair enzymes exhibit a much broader specificity that was initially expected. However, comparison of the ratio Vm rK m clearly indicates that, in fact, some of the released bases are poor substrates. This is particularly the case for DHThy for which the efficiency of excision by endo III is 10-fold lower than that of Tg. The availability of new substrates should allow a better assessment of the substrate specificity. This may provide insights into the key structural parameters required for the enzymatic excision. It would be also of interest to determine whether 5,6-dihydroxy-5,6-dihydrocytosine w64x and 5,6-dihydroxy-5,6-dihydro-5-methylcytosine w65x, two main relatively stable oxidation products of cytosine and 5-methylcytosine, respectively, are substrates for endo III and its human homologues. Another major development to be expected is the extension of the initial studies on Fpg and endo III specificity to yeast and human homologues, most of the genes of the latter proteins being recently cloned w66–69x. In this respect, it would be interesting to compare the efficiency of excision provided by both the prokaryotic and eukaryotic enzymes. It should be added that the matrix-assisted laser desorption ionization time-of-flight mass spectrometry ŽMALDI-TOF MS. technique w70,71x is a powerful tool to analyze the DNA fragments left following repair enzyme incubation. Interestingly, it was confirmed by using the latter spectrometric measurement that the AP-endonuclease activity of Fpg is able to remove abasic sites through a b–d mecha-

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nism. The situation is more confused for endo III since the mass spectrometric analysis did not provide support for the occurrence of a b-elimination process. In fact, mass fragments are rather in favor of a hydrolytic mechanism. Further work is required to resolve this apparent discrepancy. Another potential application of the MALDI-TOF method would be the analysis of the DNA–protein cross-links which result from the reduction of the Schiff base produced by the reaction of the amino acid of the active site of the enzyme with the C1 of the abasic site.

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