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PerspectiVe Bulky DNA Lesions Induced by Reactive Oxygen Species Yinsheng Wang* Department of Chemistry-027, UniVersity of California, RiVerside, California 92521-0403 ReceiVed NoVember 18, 2007
The integrity of the human genome is frequently challenged by endogenous and exogenous agents, and reactive oxygen species (ROS) constitute the major endogenous source of DNA damage. ROSinduced single nucleobase lesions have been extensively investigated; however, the formation and biological implications of bulky DNA lesions emanating from ROS exposure remain under-explored. The combination of synthetic organic chemistry and bioanalytical chemistry have led to the discovery of a group of bulky, oxidatively generated DNA lesions. In these lesions, a nucleobase, often a purine base, can be covalently bonded with the 5′ carbon of the 2-deoxyribose of the same nucleoside or its neighboring pyrimidine base to give purine cyclonucleosides and nucleobase-nucleobase intrastrand cross-links, respectively. Biochemical studies demonstrated that these lesions could markedly block DNA replication and transcription and that these lesions are repaired by the nucleotide excision repair (NER) pathway. These bulky, oxidatively induced DNA lesions may contribute significantly to neurological disorders that are associated with deficiency in NER and the natural processes of aging. Contents 1. Introduction 2. Formation of Bulky, Oxidatively Generated DNA Lesions 3. Biological Implications of Bulky, Oxidatively Induced DNA Lesions 4. Implications of Bulky, Oxidatively Generated Lesions in Human Diseases 5. Future Studies
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1. Introduction The human genome is constantly assaulted by endogenous and exogenous agents (1), among which reactive oxygen species (ROS1) can be produced by normal aerobic metabolism, by phagocytic cells in response to bacteria invasion, or by exposure to ionizing radiation and antitumoral agents (2). Under normal physiological conditions, transition metal-driven Fenton reaction constitutes the major endogenous source of ROS (3). In this regard, electrons leaking from the electron transport chain in mitochondria can combine with molecular O2 to give superoxide, which can be subsequently converted to hydrogen peroxide (H2O2) by superoxide dismutase. H2O2 is freely diffusible in the cellular environment and may reach the nucleus and interact with DNA-bound transition metal ions, iron and copper in particular, leading to the formation of highly reactive hydroxyl radical (•OH) via the Fenton-type reaction.
* To whom correspondence should be addressed. Tel: (951) 827-2700. Fax: (951) 827-4713. E-mail:
[email protected]. 1 Abbreviations: ROS, reactive oxygen species; NER, nucleotide excision repair; cyclo-dA, 8,5′-cyclo-2′-deoxyadenosine; ODN, oligodeoxyribonucleotide; XP, xeroderma pigmentosum.
Cu+/Fe2 + H2O2 f Cu2+/Fe3+ + •OH + OH-
(1)
Aside from producing •OH, the Cu2+/H2O2 system may also generate singlet oxygen, and Cu+ may promote one-electron oxidation (4–7). ROS can induce a number of covalent modifications to DNA, which encompass single-nucleobase lesions, strand breaks, inter and intrastrand cross-links, along with protein-DNA cross-links (8). In this perspective, I choose to discuss an under-investigated group of oxidatively generated DNA lesions, the bulky lesions. I will restrict my discussion to those bulky lesions that are induced from the direct interaction between DNA and ROS; bulky DNA adducts arising from lipid peroxidation products (9) will not be discussed.
2. Formation of Bulky, Oxidatively Generated DNA Lesions The interest in ROS-induced bulky lesions began with earlier studies by Randerath and co-workers (10), who detected, by using the 32P-postlabeling assay, several bulky DNA adducts that are induced endogenously in animal tissues. These adducts exhibit age-dependent increases and tissue-specific distributions (10). The bulky nature of these adducts was inferred from the chromatographic behavior of these adducts, which was similar to that of adducts formed from bulky aromatic carcinogens and the resistance of these adducts toward nuclease P1-catalyzed dephosphorylation (reviewed in ref 11). On the grounds that some bulky endogenous lesions are identical to the lesions formed in isolated DNA treated with Fenton reagents and that the levels of these lesions are elevated in kidneys of rodents treated with prooxidant carcinogens, for example, ferric nitrilotriacetate (Fe-NTA), these endogenous bulky DNA lesions were attributed to be induced by ROS (11). Moreover, some bulky adducts are commonly produced in ODNs harboring specific
10.1021/tx700411g CCC: $40.75 2008 American Chemical Society Published on Web 01/12/2008
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Chem. Res. Toxicol., Vol. 21, No. 2, 2008 277 Scheme 1. Mechanism for the Formation of the Two Diastereomers of Purine Cyclonucleosides
dinucleotide sequences, suggesting that these adducts might have the neighboring nucleobases being covalently bonded with each other (12, 13). Mutagenesis studies by using shuttle vector technology also showed that CC f TT and mCG f TT tandem double mutations could be induced by ROS generated from a variety of systems (14–17), indicating that the ROS-produced intrastrand cross-link lesions formed between neighboring nucleotides might contribute to the tandem base substitutions (14, 17). By employing ODNs housing a site-specifically inserted 8,5′-cyclo2′-deoxyadenosine (cyclo-dA), the chemical synthesis of which was realized by Cadet, Brooks, and their co-workers (18–20), Randerath et al. (21) later analyzed 32P-labeled DNA digests by thin-layer chromatography, and some of the bulky DNA adducts were assigned to cyclo-dA-carrying dinucleotides based on the coelution of these compounds with the authentic standards under nine different solvent conditions. Scheme 1 shows the mechanism for the formation of the purine cyclonucleosides. This lesion was partially resistant to cleavage with the nucleases employed in the 32P-postlabeling assay, thereby allowing for the lesion to be liberated as a dinucleotide (21). In this respect, it is worth noting that because the 32P-postlabeling method lacks structure information, more rigorous identification of endogenously induced cyclo-dA awaits future studies by using LCMS/MS. Recently, D’Errico et al. (22, 23) showed that cyclo-dA can also be detected in DNA isolated from cultured human kerotinocytes, and the level of (5′S)-cyclo-dA, as measured by using LC-MS in the selected-ion monitoring (SIM) mode, was approximately 230 lesions per 109 nucleosides (23). While the exposure of these cells to 5 Gy of X-rays results in an increase of the level of 8-oxo-7,8-dihydro-2′-deoxyguanosine by 800 lesions per 109 nucleosides (22), the same exposure leads to an elevation in the level of cyclo-dA by 50 lesions per 109 nucleosides (22, 23). However, the levels of cyclo-dA in liver DNA samples from fetal and newborn rats, as measured by the 32 P-postlabeling assay, were 15 and 27 lesions per 109 nucleosides, respectively (21), which are approximately 10 times less than what was found in cultured human cells (22, 23). Further investigation is needed to understand the origin of the marked difference in the level of the purine cyclonucleosides induced endogenously in animal tissues and formed in cultured human cells. In this respect, LC-MS operated in SIM mode offers excellent sensitivity; however, the accurate and specific detection of the low level of DNA lesions induced in cells may require more specific full-scan MS/MS or even MS3 (24–27). Despite the finding that some bulky, oxidative lesions are attributed to the cyclo-dA-bearing dinucleotides, there is continuing interest in finding ROS-induced lesions with the adjacent nucleobases in the same DNA strand being covalently joined. Earlier studies by Box et al. (28–33) demonstrated that some intrastrand nucleobase-nucleobase cross-link lesions could be induced in dinucleoside monophosphates and tetrameric ODNs upon exposure to γ- or X-rays under anaerobic condi-
Scheme 2. Formation of Secondary Radicals of Thymidine
Scheme 3. Mechanism for the Formation of G[8-5m]T and the Structures of Two Other Intrastrand Nucleobase-Nucleobase Cross-Link Lesions Discussed in This Perspective
tions. These authors further proposed that these lesions are initiated from a single hydroxyl radical attack. The hydroxyl radical either can be added to the C5 and C6 carbon atoms of pyrimidine bases or can abstract a hydrogen atom from the 5-methyl group of thymine or 5-methylcytosine (34). Both processes lead to the formation of pyrimidine basecentered secondary radicals (Scheme 2). In addition, oneelectron oxidation of thymine or 5-methylcytosine followed by deprotonation, or by both hydration and deprotonation, can also produce radicals III and II as shown in Scheme 2 (35, 36). These pyrimidine radicals may attack their neighboring purine bases to yield intrastrand nucleobase-nucleobase cross-link lesions. Scheme 3 shows the mechanism for the formation of G[8-5m]T. The above notion was later confirmed by Cadet et al. (37, 38), who demonstrated that G[8-5m]T and T[5m-8]G, where the C8 of guanine is covalently bound with the methyl carbon of its neighboring thymine, could be induced from the independently generated 5-methyl radical of thymidine. These authors further showed that these two lesions could be produced in calf thymus DNA upon exposure to γ-rays (38).
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Building upon these seminal studies, we began to systematically examine the formation and biological implications of these intrastrand nucleobase-nucleobase cross-link lesions. We first synthesized the photolabile radical precursors for the 5-methyl radical of 5-methyl-2′-deoxycytidine and the 5-hydroxy-5,6dihydro-6-yl radical of thymidine and showed that these two radicals could indeed conjugate with their neighboring guanine base to afford intrastrand cross-link lesions (39–42). It was also observed that the UVB irradiation of duplex DNA carrying a 5-bromocytosine or 5-bromouracil could lead to the covalent coupling of cytosine or uracil with its neighboring guanine or adenine to render intrastrand cross-link lesions (43–46). The above chemically synthesized intrastrand cross-link lesions facilitated the identification and quantification of these lesions induced by ROS from exposure to γ-rays or Fenton reagents. By using calf thymus DNA or synthetic doublestranded ODNs as substrates, we demonstrated that mC[5m8]G, G[8-5m]mC, and G[8-5]C (structures shown in Scheme 3) could be induced by exposure to γ-rays (41, 47). The above three lesions, along with G[8-5m]T, could also be produced in isolated DNA by Fenton reagents, that is, Cu(II)/H2O2/ascorbate or Fe(II)/H2O2/ascorbate (48, 49). Furthermore, G[8-5]C and G[8-5m]T could form in Hela cells upon exposure to γ-rays (24). We further quantified the formation of these lesions by using LC-MS/MS with the standard isotope dilution method (24, 48, 49). Several conclusions can be drawn from the quantification studies. First, the yields for the cross-link lesions are approximately 2–3 orders of magnitude lower than those for the commonly found single-nucleobase lesions (24, 25, 48, 49). Second, the formation of intrastrand cross-link lesions is sequence-dependent, with the lesion being induced more preferentially at the 5′-purine-pyrimidine-3′ site than at the corresponding 5′-pyrimidine-purine-3′ site (41, 47–49). Third, Cu(II)/H2O2/ascorbate is more than 10 times as efficient as Fe(II)/H2O2/ascorbate in inducing the intrastrand cross-link lesions (49). Last, cytosine methylation enhances, by over 10fold, the yield for the formation of intrastrand cross-link lesion (41, 49). The latter observation may have implications in the Cu(II)/H2O2/ascorbate-induced CG f TT mutation at methylated CpG sites (17).
3. Biological Implications of Bulky, Oxidatively Induced DNA Lesions The synthetic chemistry approaches also enabled researchers to obtain pure and sufficient ODN substrates housing a structurely defined lesion for examining how these lesions perturb DNA replication and how they are repaired. It was found that both diastereomers of cyclo-dA could block primer extension by mammalian and microbial replicative DNA polymerases (50, 51). In addition, cyclo-dA cannot be repaired by the base excision repair pathway (51). However, DNA substrates containing the lesion can be excised by the nucleotide excision repair (NER) activities present in nuclear extracts of wild-type (AA8) Chinese hamster ovary (CHO) cells and Hela cells, but not by the nuclear extracts of Hela or CHO cells that are deficient in some NER factors (20, 51). Host-cell reactivation assay results reveal that cyclo-dA appreciably blocks gene expression in CHO cells and human cells (20). Moreover, the bypass of cyclo-dA by human RNA polymerase II in ViVo can give rise to two types of mutant transcripts (52). In one type, the polymerase inserted uridine opposing the lesion followed by the misinsertion of adenosine opposite the template 2′deoxyadenosine immediately downstream of the lesion site. The
Wang
other type contained deletions of 7, 13, and 21 nucleotides after incorporating uridine opposite the lesion (52). The implications of cyclo-dA in the neurodegeneration of xeroderma pigmentosum patients have been discussed in detail in an excellent recent review by Brooks (53). With regard to the oxidatively induced intrastrand nucleobasenucleobase cross-link lesions, it was found that the G[8-5m]mC, G[8-5m]T, and G[8-5]C lesions could be recognized by Escherichia coli UvrABC nuclease (54, 55), supporting that these lesions might be substrates for NER enzymes in ViVo. This observation is in line with the finding that these lesions could result in considerable destabilization to the DNA double helix (54). In-Vitro replication studies showed that G[8-5m]T and G[85]C almost completely blocked the high-fidelity DNA polymerases or allowed only for the incorporation of one nucleotide opposite the 3′-thymine or cytosine portion of the two lesions (26, 56, 57). By combining shuttle vector technology with LCMS/MS, my colleagues and I recently demonstrated that the G[8-5]C cross-link blocked more than 80% of DNA replication in AB1157 E. coli cells and that it also induced considerable G f T (8.7%) and G f C (1.2%) mutations (24). Replication studies with the isogenic E. coli strains that are defective in SOS-induced bypass DNA polymerases demonstrated that pol V was responsible for the mutagenic bypass of the lesion in ViVo (24). This result is in accordance with the in Vitro replication data showing that polymerase η, the yeast homologue for E. coli pol V, is capable of synthesizing past the lesion in an errorprone fashion (47). In this context, steady-state kinetic measurement results showed that the nucleotide insertion, by yeast pol η, opposite the cytosine portion of the G[8-5]C or the thymine portion of G[8-5m]T is error-free; the polymerase inserts predominantly the correct nucleotide, dGMP or dAMP (26, 47). However, the polymerase exhibits considerable misincorporations of dAMP and dGMP opposite the guanine portion of G[85m]T or G[8-5]C (26, 47), which can lead to G f T and G f C mutations at the 5′ guanine site of the two lesions.
4. Implications of Bulky, Oxidatively Generated Lesions in Human Diseases The ROS-induced bulky lesions may have important implications in human health, including the natural processes of aging and genetic diseases associated with the deficiency in NER or the aberrant accumulations of transition metal ions. The requirement of NER for the repair of these bulky, oxidatively generated DNA lesions (20, 51, 54, 55) and the lack of NER activity in terminally differentiated cells (58, 59), particularly in neurons, may lead to the accumulation of the bulky DNA lesions in this type of cells. In this respect, it is not necessary to replicate DNA in neurons and only a small fraction of genes in them are expressed; therefore, in neurons, only those DNA lesions formed in expressed genes are repaired, whereas those generated in the bulk of the genome are not repaired (59). Decades of exposure to endogenous ROS, together with the failure in repairing the ROS-generated bulky lesions in silent genes in neurons, may bear significant implications in neurological diseases associated with aging, for example, Alzheimer’s disease (59). In this context, it was observed that in Alzheimer’s disease, the neurons that are about to die are those that have attempted to resume the cell cycle and start DNA replication (59). The accumulation of endogenous lesions in the dormant genes of neurons is likely to result in aborting the mitotic process and inducing cell death if the cells attempt to express these silent
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genes and initiate DNA synthesis. Moreover, it was observed recently that ERCC1-knockout mice exhibit a phenotype and transcription profile closely resembling those of aged mice (60), suggesting that the natural processes of aging are associated with an increased burden of ERCC1-mediated repair of bulky endogenous DNA lesions. Taken together, the accumulation of bulky, oxidatively induced DNA lesions may contribute to the natural processes of aging and aging-associated neurological symptoms. The bulky, oxidatively generated DNA lesions may play an even more significant role in neurodegeneration and cancer development in patients suffering from genetic diseases with deficiency in NER, for example, xeroderma pigmentosum (XP), trichothiodystrophy, and Cockayne’s syndrome (61, 62), because, in these patients, the repair of bulky DNA lesions are compromised even in actively transcribed genes. In this respect, XP patients exhibit markedly increased risk of developing skin cancer, which is attributed to the failure of cells to repair sunlight-induced dimeric DNA photoproducts (63). The involvement of NER in the repair of the bulky, oxidatively produced DNA lesions is consistent with clinical manifestations showing that some XP patients develop neurological abnormalities attributable to premature neuronal death (63). Neurons are not exposed to sunlight, and dimeric photoproducts cannot be induced in neurons; therefore, the neuron losses in XP patients can only be attributed to DNA lesions formed from other endogenous mutagens, for example, ROS. The ROS-induced bulky DNA lesions may accumulate in neurons of XP patients, thereby contributing to neurodegeneration in these patients (1, 64). The bulky, oxidatively generated DNA lesions may also play an important role in the pathogenesis of human diseases associated with the aberrant accumulation of iron or copper. In humans, genetic hemochromatosis and Wilson’s disease cause abnormal accumulation of iron and copper in various organs, and the formation of highly mutagenic oxidative lesions in genomic DNA has been considered to be associated with ironinduced carcinogenesis in iron-overload diseases (65). Additionally, bulky DNA lesions were found in the livers of patients with Wilson’s disease and primary hemochromatosis (66), though the chemical structures of these lesions remain undefined. Oxidatively induced bulky lesions may account, in part, for the pathological conditions of these diseases.
5. Future Studies Studies using a combination of tools in synthetic chemistry, bioanalytical chemistry, biochemistry, and molecular biology have begun to unravel the biological implications of the bulky DNA lesions induced by ROS. Future studies are needed to further illustrate the implications of this under-explored group of DNA lesions in human health. First, it is important to continue to identify the structures of bulky DNA lesions induced by ROS. It is highly likely that there are other bulky lesions of this type whose structures have yet to be elucidated, and it is possible that some of them are induced in greater quantities than the lesions discussed above. In this respect, previous studies on bulky, oxidatively induced DNA lesions have focused on ROS produced by ionizing radiation or Fenton chemistry. It is important to extend the investigation to lesions induced by other types of ROS. Along this line, approximately one-third of human cancers can be attributed to chronic inflammation (67); for example, ulcerative colitis, schistosomiasis, and atrophic gastritis were found to be closely associated with bowel, bladder, and gastric cancers,
respectively (68). It is important to assess the formation and examine the biological implications of bulky lesions induced by ROS produced during inflammation. In this context, HOCl is known to be produced in stimulated neutrophils during inflammatory responses (69–71), and recent electron paramagnetic resonance studies demonstrated that the incubation of DNA and RNA with hypochlorite can lead to the formation of exocyclic nitrogen-centered radicals of nucleobases (72, 73). These nitrogen-centered radicals may add to other nucleobases to give dimers with a radical being located on carbon atoms (72, 73). Along this line, one-electron oxidation of cytosine followed by deprotonation can lead to the formation of the exocyclic nitrogen-centered radical, which can conjugate with its vicinal cytosine to give two types of intrastrand cross-link lesions (74, 75). No intrastrand cross-link product from the reaction of HOCl with DNA or its constituents, however, has been previously isolated and structurely characterized. Nitrosoperoxycarbonate is another oxidant produced by macrophages during inflammation that can induce damage to DNA (76, 77), and it remains to be assessed whether this species can also result in the formation of the bulky lesion of DNA. Second, it is important to firmly establish the link between the bulky, oxidatively induced DNA lesions and human diseases. In this respect, it is necessary to appraise the formation of the bulky, oxidatively produced DNA lesions in tissues from patients with deficiency in NER, suffering from chronic inflammation, or bearing genetic diseases associated with the aberrant accumulations of transition metals. The outcome of these studies may lead to the discovery of biomarkers for the diagnosis and prognosis of human diseases. It is also important to identify the endogenously induced DNA lesions that are failed to be repaired in ERCC1-deficient cells. The identification of the bulky, oxidatively generated DNA lesions may further underscore their roles in the natural processes of aging and in agingrelated neurological disorders. Although the emphasis of the foregoing discussion has been placed on bulky lesions induced in the same DNA strand, recent studies revealed that ROS may also give rise to interstrand crosslink lesions. In this context, Gates and co-workers (78) showed that the abasic site induced in DNA could couple with a guanine base in the opposing strand to give an interstrand cross-link lesion. In addition, Greenberg et al. (79, 80) demonstrated that an independently generated methyl radical of thymine can conjugate with its base-paired adenine to render an interstrand cross-link product. HPLC analysis of the enzymatic digestion mixture of duplex DNA housing a [3H]-labeled thymidine suggests that the same cross-link might be induced from γ-ray exposure (81). Because the DNA interstrand cross-link prevents strand separation, the presence of these lesions in DNA is expected to markedly impede DNA replication and transcription. These lesions are, therefore, extremely cytotoxic. Further study is needed to rigorously demonstrate the formation of these lesions upon ROS exposure, especially in cells, and to assess if other types of interstrand cross-link lesions can be induced by ROS. Acknowledgment. I thank the National Institutes of Health for supporting this research (R01 CA96906 and R01 CA101864).
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