Formamidopyrimidines in DNA: Mechanisms of formation, repair, and biological effects

Free Radical Biology & Medicine 45 (2008) 1610–1621

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Free Radical Biology & Medicine j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f r e e r a d b i o m e d

Review Article

Formamidopyrimidines in DNA: Mechanisms of formation, repair, and biological effects Miral Dizdaroglu a,⁎, Güldal Kirkali b, Pawel Jaruga a a b

Chemical Science and Technology Laboratory, National Institute of Standards and Technology, Building 227/A243, Gaithersburg, MD 20899, USA Department of Biochemistry, Faculty of Medicine, Dokuz Eylul University, Izmir, Turkey

a r t i c l e

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Article history: Received 29 May 2008 Revised 26 June 2008 Accepted 8 July 2008 Available online 17 July 2008 Keywords: DNA repair Formamidopyrimidines Mutagenesis NEIL1 OH-adduct radicals Free radicals

a b s t r a c t Oxidatively induced damage to DNA results in a plethora of lesions comprising modified bases and sugars, DNA–protein cross-links, tandem lesions, strand breaks, and clustered lesions. Formamidopyrimidines, 4,6diamino-5-formamidopyrimidine (FapyAde) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua), are among the major lesions generated in DNA by hydroxyl radical attack, UV radiation, or photosensitization under numerous in vitro and in vivo conditions. They are formed by one-electron reduction of C8–OH-adduct radicals of purines and thus have a common precursor with 8-hydroxypurines generated upon one-electron oxidation. Methodologies using mass spectrometry exist to accurately measure FapyAde and FapyGua in vitro and in vivo. Formamidopyrimidines are repaired by base excision repair. Numerous prokaryotic and eukaryotic DNA glycosylases are highly specific for removal of these lesions from DNA in the first step of this repair pathway, indicating their biological importance. FapyAde and FapyGua are bypassed by DNA polymerases with the insertion of the wrong intact base opposite them, leading to mutagenesis. In mammalian cells, the mutagenicity of FapyGua exceeds that of 8-hydroxyguanine, which is thought to be the most mutagenic of the oxidatively induced lesions in DNA. The background and formation levels of the former in vitro and in vivo equal or exceed those of the latter under various conditions. FapyAde and FapyGua exist in living cells at significant background levels and are abundantly generated upon exposure to oxidative stress. Mice lacking the genes that encode specific DNA glycosylases accumulate these lesions in different organs and, in some cases, exhibit a series of pathological conditions including metabolic syndrome and cancer. Animals exposed to environmental toxins accumulate formamidopyrimidines in their organs. Here, we extensively review the mechanisms of formation, measurement, repair, and biological effects of formamidopyrimidines that have been investigated in the past 50 years. Our goal is to emphasize the importance of these neglected lesions in many biological and disease processes. Published by Elsevier Inc.

Contents Introduction . . . . . . . . . . . . . . . . . . . . . Mechanisms of formation of formamidopyrimidines . . . Formation of formamidopyrimidines in DNA in vitro and Repair of formamidopyrimidines . . . . . . . . . . . . Biological effects of formamidopyrimidines . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . Acknowledgment . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . .

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Abbreviations: FapyAde, 4,6-diamino-5-formamidopyrimidine; FapyGua, 2,6-diamino-4-hydroxy-5-formamidopyrimidine; OH, hydroxyl radical; Me-FapyGua, 2,6-diamino-4hydroxy-5N-methylformamidopyrimidine; 8-OH-Gua (8-oxo-Gua), 8-hydroxyguanine; GC/MS, gas chromatography/mass spectrometry; 2,5-FapyGua, 2,5-diamino-4-hydroxy-6formamidopyrimidine; 8-OH-Ade, 8-hydroxyadenine; Fpg, Escherichia coli formamidopyrimidine DNA glycosylase; BER, base excision repair; Nei, E. coli endonuclease VIII; Nth, E. coli endonuclease III; Ogg1, 8-oxo-Gua DNA glycosylase; hOgg1, human Ogg1; yOgg1, Saccharomyces cerevisiae Ogg1; AtOgg1, Arabidopsis thaliana Ogg1; hNEIL1, human NEIL1; mNEIL1, mouse NEIL1; T4-pdg, bacteriophage T4 pyrimidine dimer glycosylase; cv-pdg, Chlorella virus pyrimidine dimer glycosylase; COS-7, simian kidney cells; MLNE, mouse liver nuclear extracts. ⁎ Corresponding author. Fax: +301 975 8505. E-mail address: [email protected] (M. Dizdaroglu). 0891-5849/$ – see front matter. Published by Elsevier Inc. doi:10.1016/j.freeradbiomed.2008.07.004

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Introduction Oxidatively induced damage to DNA is caused when oxygenderived species such as free radicals and other oxidizing agents react with the constituents of DNA (reviewed in [1–4]). Of the free radicals, the highly reactive hydroxyl radical ( OH)1 reacts with DNA components at or near diffusion-controlled reaction rates by addition to double bonds of heterocyclic DNA bases and by abstraction of an H atom from the methyl group of thymine and each of the five carbon atoms of 2′-deoxyribose, giving rise to various OH-adduct radicals of four DNA bases, the allyl radical of thymine, and sugar radicals. Further reactions of base and sugar radicals generate a variety of modified bases and sugars, 8,5′-cyclopurine 2′-deoxynucleosides, base-free sites, strand breaks, clustered lesions, and DNA–protein cross-links [1–3]. Oxidatively induced DNA damage can be repaired by cellular repair systems using a variety of mechanisms (reviewed in [5]). If not repaired, oxidatively induced DNA damage may result in a broad range of pathophysiological processes such as mutagenesis, carcinogenesis, and aging (reviewed in [5–7]). The elucidation of the mechanisms, cellular repair, and biological consequences of this type of DNA damage is essential for an understanding of its role in disease processes. Formamidopyrimidines derived from adenine and guanine, i.e., 4,6-diamino-5-formamidopyrimidine (FapyAde) and 2,6-diamino-4hydroxy-5-formamidopyrimidine (FapyGua), respectively, are formed in DNA among the major products by various mechanisms (reviewed in [3]). Their structures as nucleosides are illustrated in Fig. 1. It should be pointed out that FapyAde and FapyGua are connected to the sugar moiety of DNA not through N1, as other pyrimidines are, but through the amino group attached to C6. These compounds have been known for 50 years as OH–induced products of adenine and guanine as free bases and nucleosides and in DNA [8–14]. Their formation in DNA under numerous in vitro and in vivo conditions and cellular mechanisms of repair has been reported (reviewed in [1–3]). Despite these facts, FapyAde and FapyGua have not received the attention in the scientific literature that they deserve, and this is the emphasis of this review. In many cases, there were misconceptions about their formation in DNA in vitro and in vivo, repair, and especially biological effects. Many misleading conclusions on repair and biological effects resulted from mistaking 2,6-diamino-4-hydroxy-5N-methylformamidopyrimidine (Me-FapyGua) for FapyGua. Results obtained with Me-FapyGua have been extrapolated to FapyGua without any data whatsoever being provided for the latter. These compounds are chemically different and are formed by distinctly different chemical mechanisms, as we will discuss below. Furthermore, much attention remains focused on another major product of guanine, i.e., 8hydroxyguanine (8-OH-Gua; also called 8-oxo-Gua) because of the relative ease of measurement and strong mutagenicity (reviewed in [15]); however, the emphasis on this specific lesion may have led to the paucity of investigation of other DNA lesions and to the detriment

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of our understanding of their biological effects. This article reviews the mechanisms of formation of formamidopyrimidines in DNA, their repair by various DNA repair enzymes and recently elucidated biological effects. The aim is to draw more attention to these important lesions, to clarify past misconceptions about them, and to emphasize their potential role in disease processes. Mechanisms of formation of formamidopyrimidines Oxidatively induced damage to DNA generates a plethora of products arising from both pyrimidines and purines and from the sugar moiety in DNA (reviewed in [1–3]). Among all these products, formamidopyrimidines are unique in that they are derived from purines and can be regarded as purines with an opened imidazole ring. However, they are pyrimidines connected to the sugar moiety of DNA not through N1, as other pyrimidines are, but through the amino group attached to C6 (Fig. 1). Mechanisms of formation of these compounds by various damaging agents have been elucidated using various techniques. The hydroxyl radical adds to the double bonds of guanine and adenine at diffusion-controlled rates with rate constants (k) of 9.2 × 109 and 6.1 × 109 M− 1 s− 1, respectively [16]. Addition of OH to the C4 and C8 positions of guanine generates C4–OH- and C8–OHadduct radicals, respectively, with possible addition to C5 generating a C5–OH-adduct radical [17–19], as illustrated in Fig. 2. The yields of the C4–OH- and C8–OH-adduct radicals comprise ∼60–70 and 17% of the intermediate radicals, respectively [19]. Reaction of adenine with OH yields C4–OH-adduct radical (50%) and C8–OH-adduct radical (37%) [18–20]. The tendency of OH addition to C5 of Ade to yield the C5– OH-adduct radical has been estimated to be less than 5% [20]. Addition of OH to C2 of both Ade and Gua has also been suggested [4,21]. Formation of 2-hydroxyadenine in DNA in vitro and in vivo is on par with this suggestion (see e.g., [21,22]). The corresponding product of Gua has not been identified. Purine OH-adduct radicals differ in their redox properties in that C4–OH-adduct radicals are oxidizing, whereas C5–OH- and C8–OHadduct radicals are reducing in nature [17,18,20]. On the other hand, these OH-adduct radicals exist in different mesomeric forms that may be oxidizing or reducing, representing a “redox ambivalence” [18–20]. C4–OH- and C5–OH-adduct radicals dehydrate (k = 5 × 103 to 1.4 × 104 s− 1), yielding a purine(-H) radical, which is reduced and protonated to reconstitute Gua or Ade [18–20,23,24]. Elimination of OH− from the Gua C4–OH-adduct radical (k = 6 × 103 s− 1) generates the guanine radical cation (Gua +), which deprotonates to give Gua(-H) (k = 1.8 × 107 s− 1) [19,25]. Gua(-H) may also be formed by dehydration of the C5–OH-adduct radical [18]. As an alternative to the addition of OH to C8, hydration (addition of OH−) of Gua + has been suggested to give rise to the C8–OH-adduct radical [26–31], although hydration may be much slower than the bimolecular decay of Gua + [19]. However, monomeric Gua + has not been found to hydrate, instead reacting with 2′-deoxyribose in DNA by H abstraction (k b 4 × 103 s− 1), leading to strand breaks [19,32]. Faster hydration of Gua + in ds-DNA than monomeric Gua + has been suggested as a possible reason for this discrepancy [19]. Using density functional theory, the H2O addition (addition of OH−) on C8 of purine radical cations (Gua + and Ade +), yielding the C8–OH-adduct radicals, has been calculated to be exothermic; however, this reaction has been found to be endothermic for the deprotonated forms Gua(-H) and Ade(-H) and thus not feasible [33]. The presence of the N1 proton of Gua + seems to be crucial for H2O addition to C8. These studies show that the C8–OHadduct radical may be formed by several mechanisms depending on reaction conditions. Without the involvement of OH, the C8–OHadduct radical may also be formed by loss of an electron from purine leading to purine +, followed by hydration (OH− addition) [27–29]. UV radiation, photosensitization, singlet oxygen, and direct effect of ionizing radiation can cause the loss of an electron from purines to give rise to purine radical cations (reviewed in [4]). This mechanism is

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Fig. 1. Structures of FapyAde and FapyGua.

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Fig. 2. Formation of OH-adduct radicals of Gua (adapted from [17] by permission of the publisher).

supported by the observed formation of 8-OH-Gua and FapyGua in DNA by photosensitization with visible light plus methylene blue or riboflavin [27,28,34–36] and by formation of FapyGua and FapyAde in DNA by UV radiation [29], as discussed below. Purine OH-adduct radicals react with oxygen at different rates. The reaction of oxygen with the Gua C4–OH-adduct radical is rather slow (k b 106 M− 1 s− 1), whereas Gua(-H) readily reacts with oxygen (k = 3 × 109 M− 1 s− 1) [19]. The latter reaction has been suggested to generate imidazolone and oxazolone derivatives [37–40]; however, pulse radiolysis experiments failed to confirm this mechanism, and a kinetically more favored alternative mechanism has been put forward

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[19]. In contrast to its Gua counterpart, the Ade C4–OH-adduct radical readily reacts with oxygen (k = 1 × 109 M− 1 s− 1) [20]. Gua and Ade C8– OH-adduct radicals react with oxygen at diffusion-controlled rates (k ≅ 4 × 109 M− 1 s− 1) [18–20]. In addition to being oxidized, they can also be reduced (Fig. 3). In competition with oxidation and reduction reactions, C8–OH-adduct radicals undergo a unimolecular opening of the imidazole ring by scission of the C8–N7 bond (k = 2 × 105 s− 1 for Gua and k = 1 × 105 s− 1 for Ade) [18–20] (Fig. 3). At low oxygen concentrations (for example 20–30 μM), the ring opening (k = 1–2 × 105 s− 1) and the reaction with oxygen (k ≅ 4 × 109 M− 1 s−1 ) may be equally efficient and thus competitive [20].

Fig. 3. Reactions of Gua C8–OH-adduct radical leading to FapyGua and 8-OH-Gua. Ade C8–OH-adduct radical follows the same reaction pattern leading to the analogous products FapyAde and 8-OH-Ade (adapted from [18] by permission of the publisher).

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One-electron oxidation of C8–OH-adduct radicals leads to the formation of 8-OH-Gua and 8-hydroxyadenine (8-OH-Ade), whereas their one-electron reduction yields FapyGua and FapyAde ([3,18]), as shown in Fig. 3 in the case of guanine. 7-Hydro-8-hydroxypurines formed by reduction without ring opening are hemiorthoamides and thus are readily converted into formamidopyrimidines (Fig. 3). Dehydration of this ring-closed form would reconstitute purine. A 1,2-H shift of the C8–OH-adduct radical, which is typical of heteroatom-centered radicals, may also contribute to the formation of both 8-OH-purines and formamidopyrimidines [4]. Both anti and syn conformations of 8-OH-Gua and FapyGua in their nucleosides have been described, but both compounds retain the anti conformation in ds-DNA [41]. Four rotameric forms of formamidopyrimidines can exist because of rotation around C5–N7 and C8–N7 bonds [42]; however, NMR studies showed that only two rotamers (cis and trans formamides) of FapyAde and FapyGua exist in solution, with the cis conformation predominating over the trans conformation [43,44]. This is also in agreement with the reported ratio of the two rotameric ring-opened forms of Me-FapyGua in poly(dGdC) [45]. However, the ratio of the two rotamers of formamidopyrimidines in DNA is not known. The ring opening of the Gua C8–OH-adduct radical followed by reduction has been studied using density functional theory [46]. The one-electron reduction of the enol form of the ring-opened C8–OHadduct radical has been proposed to follow two pathways leading to FapyGua, (a) reduction followed by tautomerization and (b) tautomerization followed by reduction, with the latter being favored over the former. Calculations using density functional theory led to the proposal of two additional pathways leading to two different formamidopyrimidine isomers [47]. The hemiorthoamide resulting from reduction of one-electron reduction of the C8–OH-adduct radical (Fig. 3) undergoes ring opening to give rise to FapyGua or to its isomer 2,5-diamino-4-hydroxy-6-formamidopyrimidine (2,5-FapyGua). The other pathway involves a proton transfer from the hydroxyl group to N7 of the C8–OH-adduct radical followed by ring opening and oneelectron reduction to lead to FapyGua or 2,5-FapyGua. This has been proposed to be the lowest energy pathway and thus may be favored over the other three pathways. 2,5-FapyGua may be converted into FapyGua via the hemiorthoamide, as there is no evidence for its formation in DNA. Relative enthalpies for these reactions have been calculated.

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them. Numerous DNA-damaging agents such as ionizing radiation, H2O2/metal ions, hypoxanthine/xanthine oxidase, photosensitization, stimulated neutrophils, UV radiation, and redox-cycling and bioreductively activated drugs have been shown to generate FapyAde and FapyGua in DNA under various experimental conditions (see e.g., [27,29,51–58]). FapyAde and FapyGua have also been identified and quantified in cultured cells and in human and animal tissues at background levels or after exposure to DNA-damaging agents or depending on pathological conditions (see e.g., [22,59–75]). As an example, Fig. 4 illustrates the levels of FapyAde and FapyGua in two control and two breast cancer cell lines at background and after exposure to H2O2, followed by a time period to allow the cells to repair DNA damage. This study showed the accumulation of FapyAde and FapyGua upon oxidative stress despite a repair period, indicating the lack of repair of these lesions in breast cancer cell lines [74]. It is noteworthy that, in most of these cases, the yield of FapyGua has been comparable or even greater than that of 8-OH-Gua. This is in contrast to the fact that 8-OH-Gua is generally presented as the “most prevalent or important product” of oxidatively induced damage to DNA in the literature, without giving any data on formamidopyrimidines or on any other lesions with comparable yields. Furthermore, it should be noted that the yields of FapyAde and 8-OH-Ade have generally been lower that those of FapyGua and 8-OH-Gua, possibly reflecting the fact that guanine is the DNA base most susceptible to oxidative damage [4,18,76,77]. In vitro studies unequivocally demonstrated that experimental conditions profoundly affect the yields of formamidopyrimidines, 8-hydroxypurines, and other modified bases. Studies have been performed using isolated DNA or chromatin and various DNA-damaging agents such as H2O2/metal ions and ionizing

Formation of formamidopyrimidines in DNA in vitro and in vivo FapyAde and FapyGua have been identified by radiation chemists as ionizing radiation-induced products of adenine, guanine, and their nucleosides and in DNA as far back as 50 years ago [8–14,48]. Addition of OH generated by interaction of ionizing radiation with water to the C8 of Gua and Ade, followed by reduction, has been proposed as a possible mechanism for the formation of formamidopyrimidines. As discussed in the previous section, these mechanisms have been confirmed by numerous studies, and perfected since then, and also by product analysis under many different reaction conditions as we will discuss below. Early studies used various chromatographic techniques to isolate and characterize formamidopyrimidines, including thinlayer chromatography, paper chromatography, ion-exchange chromatography, and high-performance liquid chromatography. The use of mass spectrometry with electron-impact coupled to gas chromatography (GC/MS) has been introduced for the measurement in DNA of formamidopyrimidines as free bases and as nucleosides [49,50]. The details of the measurement of formamidopyrimidines in DNA in vitro and in vivo will be dealt with in a separate paper (M. Dizdaroglu et al., submitted for publication). The formation of formamidopyrimidines in DNA in vitro and in vivo has extensively been studied. Because of the multiplicity of studies published on this subject, it should suffice to cite a few of

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Fig. 4. Levels of FapyAde and FapyGua in four human cell lines exposed to H2O2 and then allowed to repair DNA damage. Stars denote statistical significance compared to control (p b 0.05; n = 3). AG11134, nonmalignant human mammary epithelial cell line; HCC1937, primary ductal breast carcinoma cell line; HCC1937BL, EBV-transformed B-lymphoblastoid cell line; MCF-7, mammary epithelial adenocarcinoma cell line (from [74], reproduced by permission of the publisher). Upper graph, FapyAde; lower graph, FapyGua. Uncertainties are standard deviations.

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radiation under different gassing conditions using N2, air, N2O, and N2O/O2 (see e.g., [21,52,54,55,78,79]). The results showed that formamidopyrimidines are formed in DNA both in the absence and in the presence of oxygen, albeit with different yields, in contrast to earlier suggestions that their formation is inhibited by oxygen (see above). Oxygen, metal ions, and other oxidizing agents yielded more 8-hydroxypurines, whereas the yields of formamidopyrimidines increased in the presence of reducing agents such as ascorbic acid and glutathione. These results confirmed the mechanisms shown in Fig. 3 and lend credence to the notion that opening of the imidazole ring of the C8–OH-adduct radical competes with the reaction of this radical with oxygen as discussed above. It is important to point out that, in contrast to the formation of FapyGua as described in detail above, alkylation of guanine with compounds such as dimethyl sulfate and methylmethane sulfonate leads to 7-methylguanine, which gives rise to Me-FapyGua by opening of the imidazole ring upon treatment with alkali at high pH [45,80–85]. Me-FapyAde is also formed by alkali treatment of 7methyladenine [86]. Me-FapyGua and Me-FapyAde are chemically and mechanistically distinct from FapyGua and FapyAde and have no biological relevance in terms of oxidatively induced damage to DNA. Therefore, Me-FapyGua or Me-FapyAde should not be mistaken for FapyGua or FapyAde, respectively, in terms of mechanisms of formation, as well as in terms of repair and biological effects as we will describe below. Repair of formamidopyrimidines Formamidopyrimidines are repaired by base excision repair (BER) in living cells with the involvement of various DNA glycosylases in the first step of this repair pathway (reviewed in [87]). A DNA glycosylase that removes Me-FapyGua from DNA was first identified in extracts of various Escherichia coli strains [45,81,88,89]. This enzyme has also been found in mammalian cells [90] and then purified and named formamidopyrimidine DNA glycosylase (Fpg) [91]. Subsequently, the excision of FapyAde from γ-irradiated polydeoxyadenylic acid by this enzyme was reported [92]. The FPG gene has been cloned and Fpg has been purified to homogeneity [93,94]. Excision of both FapyAde and FapyGua by Fpg from γ-irradiated DNA containing multiple pyrimidine- and purine-derived lesions has been demonstrated [27]. Another substrate of this enzyme is 8-OHGua [27,95]. Fpg excises all three of these lesions from DNA containing multiple lesions with similar excision kinetics [96]. Despite this fact, 8-OH-Gua has wrongfully been presented in many papers as the sole physiological substrate of Fpg. This was because the main focus of interest in this field has concentrated on this lesion for some time, and because 8-OH-Gua has been the only compound measured when specificities of DNA glycosylases were investigated using oligodeoxynucleotides containing a single lesion. It is noteworthy that removal of modified bases by DNA glycosylases from oligodeoxynucleotides containing one single lesion significantly differs from excision from DNA containing multiple lesions in terms of the substrate specificities of these enzymes and excision kinetics. An extensive discussion of this phenomenon can be found elsewhere [87,97,98]. The structural basis for the recognition of FapyGua by Fpg has been elucidated, concluding that the FapyGua recognition mode of Fpg is significantly different from that of 8-OH-Gua [41]. Another study using molecular modeling and molecular dynamics simulations suggested that FapyAde and FapyGua along with 8-OH-Gua are readily bound and excised by Fpg and that these three lesions represent specific physiological substrates of Fpg [98,99], supporting the earlier suggestions [27,96]. Mutations of specific amino acids such as highly conserved Lys57, Lys155, and Pro2 have been shown to diminish or abolish the excision of FapyAde and FapyGua by Fpg [98,100,101]. Recently, oligodeoxynucleotides containing FapyAde or FapyGua at a defined position were synthesized for the first time to

enable the study of repair mechanisms and biological effects of these lesions [44,102–104]. Wiederholt et al. reported efficient excision of FapyAde and FapyGua by Fpg from these oligodeoxynucleotides [105,106], in agreement with the findings using damaged DNA containing multiple lesions as outlined above. The effect of base-pairing on the excision has been observed. Thus, Fpg removes FapyGua opposite Cyt much more efficiently than that opposite Ade, whereas FapyAde opposite all four intact bases is removed indiscriminately. In contrast to the reluctance of Fpg to excise Ade from a FapyGua–Ade pair, MutY, the other enzyme of the so-called “GO system,” has been found to remove Ade from a FapyGua–Ade mispair [106], resembling the behavior of MutY when it encounters an 8-OH-Gua–Ade mispair and removes Ade [107,108]. This is important because FapyGua, just like 8-OH-Gua, mispairs with Ade, as we will see below in more detail [109–111]. In contrast, MutY has been found to be incapable of removing Ade from FapyAde–Ade (and other intact bases) mispairs [105]. This observation suggested that organisms may not be protected from FapyAde by the GO system and there may be other repair pathways to repair this lesion. This notion is supported by efficient excision of FapyAde by E. coli endonuclease VIII (Nei) from FapyAde– Ade mispair in oligodeoxynucleotides and exclusive removal of FapyAde and FapyGua from DNA by human and mouse NEIL1 proteins (homologues of Nei), as we will discuss below. FapyAde and FapyGua are also substrates of Fpg of the ionizing radiation-resistant bacterium Deinococcus radiodurans [112]. The E. coli DNA glycosylases endonuclease III (Nth) and Nei are mainly specific for pyrimidine-derived lesions (reviewed in [87]). However, FapyAde is also efficiently removed, but not FapyGua, by these enzymes from DNA with excision kinetics similar to those of pyrimidine-derived lesions [113,114]. Experiments with synthetic oligodeoxynucleotides containing either FapyAde or FapyGua showed that Nei efficiently excises FapyAde opposite Ade or Thy, with the excision from the FapyAde–Ade pair being more significant than that from the FapyAde–Thy pair [115]. Excision of FapyGua opposite Ade was much less efficient than that of FapyAde opposite Ade. Moreover, excision from the native FapyGua–Cyt pair was very low. A similar trend has been observed with more efficient excision of FapyAde than of FapyGua by Nth from synthetic oligodeoxynucleotides [115]. In fact, FapyGua paired with pyrimidines was a very poor substrate for Nth. These results are in excellent agreement with those showing that Nth and Nei efficiently excise FapyAde, but not FapyGua, from DNA containing multiple lesions [113,114]. Many eukaryotic DNA glycosylases are also specific for FapyAde and FapyGua. Saccharomyces cerevisiae 8-oxo-Gua DNA glycosylase (yOgg1) possesses substrates in common with E. coli Fpg, although no significant homology exists between them [116]. yOgg1 excises 8-OHGua and Me-FapyGua from oligodeoxynucleotides and FapyGua and 8OH-Gua, but not FapyAde, from DNA containing multiple lesions [117]. Similar specificities have been observed with human Ogg1 (hOgg1), Drosophila melanogaster Ogg1, and Arabidopsis thaliana Ogg1 (AtOgg1) [118–121]. It should be pointed out that the main difference between bacterial Fpg proteins and eukaryotic Ogg1 proteins is the lack of excision of FapyAde by the latter. In agreement with this fact, a recent study showed that hOgg1 efficiently excises FapyGua, especially when correctly paired with Cyt, from synthetic oligodeoxynucleotides and that, in contrast, FapyAde is excised somewhat when mispaired with Cyt only [122]. Mutations in human OGG1 leading to single amino acid substitutions have been shown to significantly affect the specificity of Ogg1 for FapyGua and 8-OH-Gua. Thus, the polymorphic α-Ogg1Cys326 possesses a lower activity for both FapyGua and 8-OH-Gua than the wild-type α-Ogg1-Ser326 [118]. This is also true for two other mutant proteins, α-Ogg1-Gln46 and α-Ogg1-His154 [119], which had been found in cancerous tissues [123,124]. Another protein discovered in D. melanogaster and named protein S3 is located to ribosomes and excises FapyGua and 8-OH-Gua, but not FapyAde [125], in analogy to eukaryotic Ogg1 proteins.

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S. cerevisiae Ntg1 and Ntg2 proteins, whose amino acid sequences are closely related, have been shown to excise FapyAde and FapyGua, but not 8-OH-Gua, along with a number of modified pyrimidines from DNA containing multiple lesions [126]. Kinetic parameters for excision of FapyAde and FapyGua by both enzymes were similar to one another and also to those for excision of most modified pyrimidines. In agreement with these findings, Ntg1 has been found to efficiently remove both FapyAde and FapyGua, but not 8-OH-Gua, from synthetic oligodeoxynucleotides [122,127]. FapyGua is efficiently removed by Ntg1 from its pairs with Cyt or Ade, whereas the identity of the opposite base does not affect the excision of FapyAde [122]. These findings clearly suggest that one of the biological roles of these yeast enzymes is to remove formamidopyrimidines from damaged DNA. Table 1 shows the specificity constants for excision of FapyAde, FapyGua, and 8-OH-Gua by prokaryotic and eukaryotic DNA glycosylases that have been measured by GC/MS under identical conditions. A compilation of specificity constants for excision by DNA glycosylases of oxidatively induced lesions from DNA can be found elsewhere [87]. These data strongly suggest that both FapyAde and FapyGua are physiological substrates of most of the known prokaryotic and eukaryotic DNA repair enzymes. Furthermore, they show that both FapyGua and 8-OH-Gua are excised with similar excision kinetics by Ogg1 proteins and suggest that these lesions are the physiological substrates of Ogg1 proteins, in contrast to most work in the literature presenting the latter as the sole substrate. It should be pointed out that no significant excision of formamidopyrimidines has been observed by Schizosaccharomyces pombe Nth or by human NTH1 from DNA containing multiple lesions [128,129]. Recently, the repair of FapyAde and FapyGua has been investigated using mitochondrial and nuclear extracts from wild-type mice and knockout mice lacking Ogg1 or NTH1 or both [70]. Extracts from wildtype mice efficiently removed both FapyAde and FapyGua from synthetic oligodeoxynucleotides. Fig. 5 illustrates these results obtained with mouse liver nuclear extracts (MLNE). In addition, nth1−/− mouse extracts removed FapyGua, but failed to remove FapyAde, providing the evidence that FapyAde is a substrate of mouse NTH1, but not FapyGua. In contrast, ogg1−/− mouse extracts removed FapyAde, but not FapyGua, proving that the former is not a substrate of mouse Ogg1, but the latter is. As expected, doubleknockout mouse (ogg1−/−/nth1−/−) extracts removed none of these lesions. Moreover, accumulation of FapyAde has been observed in liver DNA of nth1−/− mice, but not in that of ogg1−/− mice compared with wild-type mice. FapyGua accumulated in liver DNA of ogg1−/− mice. Mitochondrial extracts yielded similar results. These findings are in excellent agreement with the specificities of other eukaryotic Ogg1 proteins elucidated using pure enzymes, damaged DNA, and oligodeoxynucleotides, as discussed above. Another class of DNA glycosylases has recently been discovered in humans and mice and named NEIL1 because of its sequence similarity to E. coli Nei [130–135]. A unique feature of NEIL1 is that its activation depends on cell cycle and it is expressed in cells predominantly during the S phase [131]. This indicates that the function of NEIL1 is likely to be associated with replication [136,137]. The structure of human NEIL1 (hNEIL1) has been solved using X-ray crystallography [138]. This enzyme does not contain a zinc-finger motif like E. coli Fpg and Nei,

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Fig. 5. Incision of oligodeoxynucleotides at FapyAde or FapyGua residues by liver nuclear extracts (MLNE) from wild-type and knockout mice (from [70], reproduced by permission of the publisher). Uncertainties are standard deviations.

but it maintains a “zinc-less finger” motif. When oligodeoxynucleotides with one single lesion were used, both hNEIL1 and mouse NEIL1 (mNEIL1) excised Me-FapyGua and a number of pyrimidine-derived lesions, but not 8-OH-Gua paired with Cyt [130–132,134,136]. In contrast, one paper reported the excision of 8-OH-Gua [133], which has not been confirmed later by others. On the other hand, human and murine NEIL1 proteins have been demonstrated to efficiently excise the secondary products of 8-OH-Gua, namely guanidinohydantoin and the two diastereomers of spiroiminodihydantoin from oligodeoxynucleotides [139,140]. It is not known, however, whether these lesions are relevant substrates of these enzymes in vivo or play a role in disease processes observed in neil1−/− animals. When DNA containing multiple lesions was used as a substrate, hNEIL1 efficiently excised FapyAde and FapyGua only [131,141]. Again, 8-OH-Gua was not a substrate. So far, hNEIL1 is the only known human enzyme that excises FapyAde. Similar to its human functional homologue, mNEIL1 exhibited an efficient activity for FapyAde and FapyGua, but not for 8-OH-Gua, and some minor activity for the pyrimidine-derived lesions thymine glycol and 5-hydroxy-5-methylhydantoin [142]. Fig. 6 illustrates the excision of FapyAde and FapyGua by mNEIL1 from DNA containing multiple lesions. These data unequivocally show the lack of excision of 8-OH-Gua from DNA

Table 1 Comparison of the specificity constants [kcat/KM × 105 (min −1 nM −1)]a for excision of FapyAde, FapyGua, and 8-OH-Gua from DNA with multiple lesions by E. coli Fpg, Nth, and Nei [96,113,114]; yOgg1 [117]; hOgg1-Ser326 and hOgg1-Cyt326 [118]; AtOgg1 [121]; Ntg1 and Ntg2 [126]; and mNEIL1 [142] Substrate FapyAde FapyGua 8-OH-Gua a b

Fpg 17.6 ± 1.2 25.7 ± 2.9 13.0 ± 1.3

Nth 3.1 ± 0.3 —b —b

Nei 5.0 ± 0.3 —b —b

yOgg1 b

— 1.5 ± 0.1 6.5 ± 0.4

Values represent the mean ± standard deviation (n = 6). No excision detected, or excision was too low for determining excision rates.

hOgg1-Ser326 b

— 9.0 ± 0.2v 4.5 ± 0.1

hOgg1-Cyt326

AtOgg1

Ntg1

Ntg2

mNEIL1

—b 4.4 ± 0.1 2.8 ± 0.1

—b 6.6 ± 0.2 —b

4.9 ± 0.7 3.6 ± 0.5 —b

4.7 ± 0.5 2.4 ± 0.6 —b

7.1 ± 0.1 9.3 ± 0.6 —b

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activity, these structurally similar (41% identity) enzymes have been found to efficiently catalyze excision of FapyAde and FapyGua from UV- or γ-irradiated DNA [152,153]. T4-pdg exhibited an N-glycosylase activity for FapyAde, but did not significantly excise FapyGua. The rate of this activity has been estimated be ∼1% of that for the incision at pyrimidine dimers. On the other hand, cv-pdg efficiently excised both FapyAde and FapyGua as an N-glycosylase. Its activity for FapyAde was approximately fourfold greater than that for FapyGua. Furthermore, cv-pdg was approximately threefold more efficient than T4-pdg for excision of FapyAde. The fact that UV radiation produces FapyAde and FapyGua in DNA [29], as discussed above, means that the viral enzymes T4-pdg and cv-pdg are not only specific for pyrimidine dimers, but also possess an N-glycosylase activity for UV radiationinduced monomeric formamidopyrimidines in DNA. Biological effects of formamidopyrimidines Fig. 6. Excision of FapyAde and FapyGua by mNEIL1 from DNA with multiple lesions. The dependence of excision on the enzyme amount is shown (from [142], reproduced by permission of the publisher). Uncertainties are standard deviations.

containing multiple lesions by mNEIL1. The specificity constants for excision of FapyAde and FapyGua by mNEIL1 are given in Table 1. There is evidence that, unlike other DNA glycosylases, for example, Ogg1 and NTH1, specific for repair in double-stranded DNA substrates, NEIL1 is preferentially involved in repair of lesions in single-stranded DNA fork and bubble structures generated during replication and/or transcription [137,143–145]. Recent data obtained with neil1−/− mice strongly suggest that NEIL1 may play a significant role in the prevention of the diseases associated with metabolic syndrome and cancer [146]. A correlation of inactivating mutations in neil1 with human gastric cancer has also been demonstrated [147]. Moreover, significant reduction in the mRNA levels of neil1 sensitized cells to the killing effects of ionizing radiation [135]. These findings lend credence to the importance of FapyAde and FapyGua in disease processes. In support of this notion, significant accumulation of FapyAde and FapyGua, but not 8-OH-Gua, has been observed in neil1−/− mice, who exhibited late onset of cancer (G.W. Teebor et al., submitted for publication). Moreover, four polymorphic variants of hNEIL1 have recently been isolated, S82C, G83D, D252N, and C136R [141]. S82C, G83D, and D252N retained near-wild-type activity on abasic sitecontaining oligodeoxynucleotides, although G83D failed to catalyze the wild-type β,δ-elimination reaction, but yielded the β-elimination product. The abasic site-nicking activity of C136R was significantly reduced. S82C and D252N exhibited near-wild-type glycosylase activity for excision of FapyAde and FapyGua from damaged DNA, whereas G83D lacked this activity entirely. These findings suggest that individuals with inactive neil1 alleles may be at risk for disease development and that formamidopyrimidines may play a role in these processes. Furthermore, the decrease in the level of NEIL1 led to accumulation of oxidatively induced DNA damage and enhanced spontaneous mutations in the Hprt locus in human and Chinese hamster cells [137]. Mutations have been further enhanced under oxidative stress. The majority of mutations (75–80%) in the Hprt locus have been found at Ade/Thy pairs, indicating that lesions derived from Ade and/or Thy are preferred substrates of NEIL1. This finding is consistent with the substrate preference of NEIL1 for excision from DNA containing multiple lesions in terms of FapyAde and Thy-derived lesions as described above [131,135,141]. The number of the mutations at Ade/Thy pairs was significantly greater than that at Gua/Cyt pairs. The low level of mutations at Gua/Cyt pairs may have resulted from the efficient excision of FapyGua and 8-OH-Gua by Ogg1, which is not specific for FapyAde (see above). Viral DNA glycosylases such as bacteriophage T4 pyrimidine dimer glycosylase (T4-pdg) and Chlorella virus pyrimidine dimer glycosylase (cv-pdg) are specific for removal of UV radiation-induced cyclobutane pyrimidine dimers from DNA [148–151]. In addition to this major

In early studies, the biological effects of formamidopyrimidines have been mainly inferred from the studies done with Me-FapyGua, which results from methylation of Gua and subsequent treatment with alkali as discussed above. These studies showed that MeFapyGua blocks DNA chain elongation by E. coli DNA polymerase, but does not mispair with either Ade or Thy, suggesting that Me-FapyGua is a lethal lesion rather than a mutagenic one [154]. Subsequent studies showed that E. coli DNA polymerase Klenow fragment and T4 polymerase are inhibited one base 3′ of template Ade and Cyt residues [83] and that Me-FapyGua inhibits DNA synthesis in E. coli, indicating its killing potential [84,155]. A recent study also showed Me-FapyGua to be a fairly strong but not absolute block to DNA synthesis, thus being potentially lethal, but not a mutagenic lesion [85]. In the same context, FapyAde has been suggested to be a weak inhibitor of DNA synthesis by prokaryotic DNA polymerases, depending on the sequence context and DNA polymerase [156]. FapyGua may possess similar properties. Some mutagenic properties have been attributed to Me-FapyGua leading to G → T and G → C transversions [84,86]. However, this potency is very low and DNA polymerases inserted mainly the correct base, Cyt, opposite Me-FapyGua [85,86]. In contrast, Me-FapyAde seemed to be a potent miscoding lesion, mispairing with Cyt, leading to A → G transitions, although this lesion cannot assume a Gua-like hydrogen bonding pattern [86]. Possible mutations caused by FapyAde and FapyGua have been investigated in M13pm18 phage DNA damaged by hypoxanthine/xanthine oxidase/Fe (III)–EDTA and then transfected to SOS-induced and uninduced E. coli [157]. This DNA-damaging system abundantly produces FapyAde and FapyGua in DNA among other lesions [51]. An increase in A → G transitions has been observed and attributed to FapyAde, suggesting incorporation of Cyt opposite this lesion with a Gua-like hydrogen binding [86,157]. However, no guanine-derived mutations have been observed, pointing to FapyGua as a lethal lesion similar to MeFapyGua. Despite these efforts, no unequivocal conclusions could be drawn about the biological effects of FapyAde and FapyGua. This was due to the lack of oligodeoxynucleotides with a single FapyAde or FapyGua inserted at a defined position. In contrast, oligodeoxynucleotides with a single lesion inserted at a defined position have been used for the past 2 decades to investigate the mutagenic properties of 8OH-Gua, 8-OH-Ade, and other lesions (see e.g., [158–167]). Recently, such oligodeoxynucleotides containing FapyAde or FapyGua at a defined position have been synthesized for the first time [44,102–104]. This enabled the study of base-pairing properties and biological effects of both FapyAde and FapyGua. Using these oligodeoxynucleotides, FapyAde has been found to direct Klenow exo− fragment from E. coli DNA polymerase I to preferentially misincorporate Ade opposite itself with a hydrogen bonding pattern that involves the syn conformation of the β isomer of the nucleoside form [168]. Mispairing of FapyAde with Ade suggests that it will lead to A → T transversions. This finding is in contrast to that suggesting the incorporation of Cyt

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opposite FapyAde when it was generated randomly in DNA, possibly leading to A → G transitions [86,157]. Moreover, FapyAde may be a more potent premutagenic lesion than 8-OH-Ade [168], which has the precursor Ade C8–OH-adduct radical in common with FapyAde (see above). Similarly, Klenow exo− misincorporates Ade opposite FapyGua, resulting in G → T transversions [109]. This is the same type of mutation caused by 8-OH-Gua (reviewed in [15]). Mispairing of FapyGua with Ade has been rationalized using two syn conformational isomers, with the likely involvement of the β isomer of the nucleoside form, with one of them being analogous to 8-OH-Gua–Ade pairs [169] and the other one having a Thy-like hydrogen bonding pattern. The hydrogen bonding pattern presented by FapyGua was significantly different from that of Me-FapyGua, confirming important differences between these compounds as indicated above. These findings suggested that the bypass by Klenow exo− of FapyGua with misincorporation of the wrong base is much more extensive than that of Me-FapyGua, FapyAde, and 8-OH-Ade, but it is comparable to the bypass of 8-OH-Gua [109]. A subsequent study compared the mutagenesis caused by formamidopyrimidines and 8-OH-purines in simian kidney cells (COS-7) [110]. Significant misincorporation opposite FapyGua occurred, inducing G → T transversions, but this depended significantly on the sequence context. 8-OH-Gua also led to G → T transversions in the same sequence context as FapyGua, but FapyGua was about 25–35% more mutagenic than 8-OH-Gua. In contrast, FapyAde and 8-OH-Ade have been determined to be weakly mutagenic depending on the sequence context, leading to A → C transversions. Molecular modeling studies provided a possible explanation for the sequence-context dependency. In contrast to the finding in COS-7 cells, a recent study showed that FapyGua is weakly mutagenic when bypassed in E. coli in different sequence contexts [111]. This study also determined the mutagenicity of 8-OH-Gua in E. coli and found it to be up to approximately fivefold greater than that of FapyGua. The findings discussed above unequivocally show that the biological effects of FapyAde and FapyGua almost completely differ from those of methylated formamidopyrimidines. Thus, these findings correct misconceptions and conclusions previously presented in the literature on the biological effects of formamidopyrimidines without any data on them provided. Furthermore, it is now well known from these studies and others discussed above that FapyGua is formed as abundantly and is at least as mutagenic as 8-OH-Gua, if not more so, in mammalian cells. These facts should also correct a misconception in the literature that 8-OH-Gua was the most prominent and most mutagenic lesion formed in DNA by oxidatively induced damage. There exist numerous papers in the literature on the involvement of human ogg1 mutations in different types of human cancer. The ogg1 gene has been localized to chromosome 3p25, which frequently exhibits loss of heterozygosity in many types of tumors [170]. Due to a genetic polymorphism at codon 326 (Ser326Cys), two forms of Ogg1, α-Ogg1-Ser326 and α-Ogg1-Cys326, are produced in human cells [171]. The polymorphic form has been found in the Japanese population in both healthy individuals (up to 47%) and lung and gastric cancer patients, and in European patients with head, neck, and kidney tumors [123,171–173]. A greater activity of α-Ogg1-Ser326 compared to αOgg1-Cys326 was observed in the complementation assay of an E. coli mutant defective in the repair of 8-OH-Gua [171]. Another mutant, αOgg1-Gln46, exhibited the same effect. Numerous subsequent studies reported the association of the Ser326 → Cys326 mutation with the risk of esophageal, colon, orolaryngeal, lung, gastric, cervical, gallbladder, and bladder cancers [174–185]. Mutations in ogg1 have also been found in a significant fraction of patients with Alzheimer's disease [186]. Low Ogg1 activity has been associated with an increased risk of lung, head, and neck cancers [187,188]. An assay has been developed to measure the Ogg1 activity in human tissues to determine this type of association [189]. The studies discussed above almost exclusively dealt with 8-OH-Gua as the only substrate of Ogg1 and the

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conclusions have been drawn accordingly. However, as we discussed above and previously (reviewed in [87]), eukaryotic, including human, Ogg1 proteins are specific not only for 8-OH-Gua, but also for FapyGua. The polymorphic α-Ogg1-Cys326 exhibits a lower activity for both 8OH-Gua and FapyGua than the wild-type α-Ogg1-Ser326 [118] (Table 1). This is analogous to the reported lower activity of the former compared with the latter in the complementation assay of an E. coli mutant defective in the repair of 8-OH-Gua [171]. Two mutant enzymes, α-Ogg1-Gln46 and α-Ogg1-His154, which have been identified in human cancerous tissues [123,124], possess a lower activity for excision of 8-OH-Gua and FapyGua from DNA than α-Ogg1-Ser326 [119]. This indicates that the single amino acid substitutions significantly affect the specificity of hOgg1 for these two lesions. These findings unequivocally show that FapyGua is as important a substrate of human Ogg1 proteins as 8-OH-Gua. Therefore, conclusions that have been drawn from the aforementioned studies on the involvement of human ogg1 mutations in different types of human cancer with the emphasis on 8-OH-Gua may also be true for FapyGua, meaning that the latter also plays an important role in carcinogenesis. Overall, all the facts discussed in this section strongly suggest that formamidopyrimidines may play an important role in the mutagenic effects of oxidatively induced damage to DNA and in various disease processes. Conclusions Formamidopyrimidines are unique purine-derived lesions with an open imidazole ring generated by hydroxyl radical, UV radiation, and photosensitization in vitro and in vivo under a variety of conditions. These lesions and 8-OH-purines are produced from a common precursor upon one-electron reduction and one-electron oxidation, respectively. In many cases, the background levels and yields of formamidopyrimidines are comparable to those of other major DNA lesions formed by oxidatively induced damage, including 8-OH-Gua, the most investigated lesion. There are mass spectrometric techniques to accurately measure these lesions in DNA in vitro and in cultured cells and tissues in vivo. A large number of prokaryotic and eukaryotic DNA glycosylases with different substrate specificities exist for efficient removal of formamidopyrimidines from DNA in the BER pathway. DNA glycosylases that efficiently remove 8-OH-Gua are equally specific for formamidopyrimidines. However, there are those enzymes that efficiently excise these lesions from DNA, but exhibit no specificity for 8-OH-Gua. Recent studies unequivocally proved that FapyGua and FapyAde possess mutagenic properties albeit with different extents, contrasting with previous conclusions drawn from the work on their methylated analogues. The formation and accumulation of formamidopyrimidines under numerous pathological conditions attest to their importance in disease processes. More work is warranted to further understand the biological effects of these important DNA lesions of oxidatively induced damage to DNA. Acknowledgment This paper is dedicated to the memory of Dr. Ewa Gajewski, an excellent friend and collaborator. References [1] Dizdaroglu, M. Oxidative damage to DNA in mammalian chromatin. Mutat. Res. 275:331–342; 1992. [2] Breen, A. P.; Murphy, J. A. Reactions of oxyl radicals with DNA. Free Radic. Biol. Med. 18:1033–1077; 1995. [3] Evans, M. D.; Dizdaroglu, M.; Cooke, M. S. Oxidative DNA damage and disease: induction, repair and significance. Mutat. Res. 567:1–61; 2004. [4] von Sonntag, C. Free-Radical-Induced DNA Damage and Its Repair. Springer, Heidelberg; 2006. [5] Friedberg, E. C.; Walker, G. C.; Siede, W.; Wood, R. D.; Schultz, R. A.; Ellenberger, T. DNA Repair and Mutagenesis. ASM Press, Washington, DC; 2005.

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