DNA Damage: Types of DNA Damage

Exogenous DNA Lesions

DNA lesions induced by exogenous factors are multifaceted and depend on the quality of the agent and the target on which it acts. Such lesions comprise the exogenous UV-induced formation of pyrimidine dimers on the one hand, and, on the other hand, the formation of DNA strand breaks and adducts, base alkylation, and tautomeric isomerism of nitrogenous bases, to name but a few. Previous investigations identified the DNA-damaging potential of UV-C as being mediated by covalently linking two adjacent pyrimidines that belong to the same strand of DNA or to different DNA strands. This dimerization process predominantly generates cyclobutane pyrimidine dimers and pyrimidine (6 – 4) pyrimidone photoproducts (Figure 1) ^2-8. In cyclobutane pyrimidine dimers, the two adjacent pyrimidines are covalently connected by a cyclobutane ring whereas, in pyrimidine (6 – 4) pyrimidone photoproducts, the C6 position of one pyrimidine is covalently attached to the C4 position of the neighboring pyrimidine. Accumulation of cyclobutane pyrimidine dimers and pyrimidine (6-4) pyrimidone photoproducts can lead to cell death or mutagenesis.  UV-A and UV-B are also capable of inducing the formation of pyrimidine dimers, but to a much lesser extend ^{1, 4}. UV-A has also been found to damage DNA by causing formation of DNA adducts through photo-oxidation reactions and the excitation of endogenous (e.g., flavins, porphyrins) and exogenous  photosensitizers (e.g., methylene blue, promazine, psoralens, tetracycline) ^9-12. Particularly, UV-A-mediated photosensitization has been shown to induce the synthesis of the highly mutagenic 8-oxo-7,8-dihydroguanine (8-oxo-G) via formation of singlet oxygen (¹O₂) or a type I photosensitization reaction ^{9, 13-15}. It is well known that, in mammalian cells, near and far UV radiation causes DNA protein crosslinking, whereas UV-A radiation drives the formation of DNA strand breaks ^{16, 17}.

Figure 1. UV-induced pyrimidine dimers in DNA. (Left) Cyclobutane pyrimidine (thymine-thymine) dimer; (Right) pyrimidine (6 – 4) pyrimidone photoproduct. Image was reproduced, with permission, from Nikitaki et al. (2015) ⁶⁴⁴. License at https://creativecommons.org/licenses/by/4.0/

Environmental factors such as polycyclic aromatic hydrocarbons (PAH) also induce the formation of bulky adducts in DNA that may block DNA polymerase II at the lesion site and, in turn, afflict DNA repair mechanisms which may induce tumorigenesis ¹⁸. PAH can be found not only in automobile exhaust and tobacco smoke, but also in charred food and incomplete incineration of fossil fuels and organic material ^{19, 20}. Examples of PAH include anthracene, naphthalene, pyrene, 1-hydroxypyrene, 1-nitropyrene, benzo[a]pyrene and dibenzo[a,l]pyrene. PAH activate the cytochrome P450 system in the liver and produce reactive compounds interacting with DNA ²¹ . A common and well studied adduct-forming agent is benzopyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), which is produced in vivo from benzo[a]pyrene in tobacco smoke (Figure 2). With respect to carcinogenicity, dibenzo[a,l]pyrene functions as the most effective PAH and thus represents a major cancer risk factor to humans ²².

 

Figure 2: Adduct formation between guanine (A) or adenine (B) and benzopyrene-7,8-dihydrodiol-9,10-epoxide (BPDE). Image was reproduced from Muñoz and Albores (2011) ⁶⁴⁵. License at https://creativecommons.org/licenses/by/3.0/.

 

Apart from polycyclic aromatic hydrocarbons (PAH), DNA damage is also caused by further exogenous factors, including alkylating agents, aromatic amines, reactive electrophiles, toxins as well as environmental stresses (for a review see Chatterjee and Walker, 2017 ⁵; Chakarov et al., 2014 ¹). Exogenous alkylating substances are commonly generated from dietary components, biomass combustion, tobacco smoke, industrial processing and chemotherapeutics ^23-25. Amongst them, haloalkanes (dichloromethane, trichloromethane, tetrachloromethane), alkyl sulfonates, and nitrosoureas are the most common alkylating compounds. Additional alkylating agents encompass sulfur and nitrogen mustards that gained questionable fame as mustard gas in World War I and II. Alkylating agents add the alkyl group by, either, i) an S_N1 substitution reaction which proceeds via the first order kinetics and exhibits a carbonium ion intermediate, or, ii) an S_N2 substitution reaction which proceeds through the second order kinetics, generally forming adducts with less mutagenic and carcinogenic potential than those of the S_N1 pathway ^{5, 26, 27}. The mustards have been characterized by the presence of two reactive moieties enabling the interaction with two distinct sites in the DNA. These bifunctional activities culminate in the formation of intra- and interstrand crosslinks on the one hand and, on the other hand, in the generation of DNA-protein crosslinks that inhibit DNA reactivity ^{28, 29}. Some of the most potent and widely used anti-cancer compounds comprise cyclophosphamide ³⁰, dacarbazine ³¹ and cisplatin ³²; all of them working by alkylation of DNA.

 

Aromatic amines represent a further class of alkylating agents that are activated and converted into carcinogenic mediators by the P450 mono-oxygenase system. Such aromatic amines encompass, amongst others, 2-aminofluorene (AF) and its acetylated derivative N-acetyl-2-aminofluorene (AAF), interacting with the guanine at position C8 ^{26, 33}. C8-guanine alkylation by aminofluorenes is documented to form steady lesions leading to base substitutions and frameshift mutations as the basis of its mutagenic capacity ^34-36. Covalent DNA adducts are also generated by the reactive electrophile, 4-nitroquinoline 1-oxide (4NQO1) which harbors mutagenic and carcinogenic potential ⁵. 4NQO1 is metabolized to 4-acetoxyaminoquinoline 1-oxide (Ac-4HAQO) and subsequently forms covalent adducts with C8 or N2 of guanine and N6 of adenine, respectively. It also causes oxidative stress that leads to the generation of 8-hydroxyguanine, all of which contribute to DNA strand breakage and tumorigenesis (for details see Chatterjee and Walker, 2017) ⁵.

 

Environmental stressors (cold, heat, oxidative stress, hypoxia) and natural toxins are also capable of inducing DNA damage. The group of John Wilson convincingly demonstrated that environmental stress induces trinucleotide repeat mutagenesis by alternative non-homologous end joining (NHEJ) repair, thereby contributing to genetic mutability in cancer cells ^{37, 38}. Aflatoxin B1 from Aspergillus spec., a most powerful liver carcinogen, has been found as being metabolized via the P450 mono-oxygenase system into a reactive electrophile that interacts with the N7 of guanine to generate 8,9-dihydro-8-(N7-guanyl)-9-hydroaflatoxin B1, which impairs the glycosidic linkage and, in turns, causes depurination of the DNA ^{39, 40}. Moreover, certain ingredients of a wide range of everyday articles have been noted to cause DNA damage such as food additives (e.g., citric acid, phosphoric acid) and preservatives (e.g., potassium and sodium benzoate) as well as constituents of cosmetics, pharmacons, and comestible goods such as butyl paraben and bisphenol A (for a review see Chatterjee and Walker, 2017) ⁵.

 

Exogenous DNA damage is also induced by ionizing radiation (IR) which has always been a natural component of our environment. IR is able to attack DNA directly by dissolving its molecular architecture (physical breakage of the phosphodiester bond in the DNA backbone, destruction of deoxyribose ⁴¹) or indirectly by, for instance, water radiolysis leading to the formation of highly reactive oxygen species (ROS) such as hydroxyl (HO•) and alkoxy (RO₂•) radicals ⁴². H₂O₂ which is generated by disproportionation of superoxide radicals also attacks DNA, thereby attenuating cellular functions or inducing cell death ⁴³. Actually, the majority of IR-induced DNA damage is based on the indirect action mechanism predominantly mediated by HO• radicals (Figure 3) ⁴⁴. HO• can interact wit the C5 or  the carbon atom from the methyl group (CH₃) abundant in the pyrimidines, which is in accordance with the C8 in purines or the amino group of adenines ⁴⁵. DNA damage may also be induced by reactive nitrogen species (RNS) as a result of the ionization of DNA atoms ⁴³. Collectively, IR produces a broad spectrum of base modifications such as 8-oxo-guanine, thymine glycol, and formamidopyrimidines ⁵. Amongst them, 8-oxo-guanine (8-oxo-7,8-dihydroguanine) represents the predominant oxidative DNA lesion in the human genome ⁴⁶.

 

Figure 3: Ionizing radiation acts directly and indirectly on DNA. Image was reproduced, with permission, from Desouky et al. (2015) ⁴². License at https://creativecommons.org/licenses/by-nc-nd/4.0/

 

Beside its potential to modify nucleic acid bases, IR produces a wide diversity of DNA lesions amongst which double-strand breaks (DSB) are considered as being the central player crucially involved in cell death and genomic destabilization. If left unrepaired or inadequately repaired, DSB give rise to chromosomal aberrations as being a possible source for several human diseases ⁴⁷. Of note, IR can also lead to tautomeric isomerization of nitrogenous bases (Figure 4) which might affect the nucleotide sequence in the DNA or its structural characteristics, thus culminating in structural or functional alterations possibly associated with pathological changes ¹.

Figure 4: Tautomerisation of nitrogenous DNA bases. Image was reproduced, with permission, from Chakarov et al. (2014) ¹. License at https://creativecommons.org/licenses/by/4.0/

 

Endogenous DNA Lesions

 

Spontaneous base deamination, DNA methylation, depurination/depyrimidination

Spontaneous deamination of nitrogenous bases represents a common type of hydrolytic DNA cleavage and the dominant cause of spontaneous mutagenesis in human cells. Base deamination leads to the loss of the exocyclic amine culminating in base conversion. As summarized by Chatterjee and Walker (2017) ⁵, adenine is converted to hypoxanthine while deamination of cytosine forms uracil. Cytosine and 5-methyl cytosine are the most commonly deaminated bases, but deamination of 5-methyl cytosine occurs three to four times more often than cytosine deamination and results in the production of thymine. Deamination of 5-methyl cytosine within CpG islands, regions with high CG content in the promoter region of genes, results in a mismatched pair of T/G, serving as a substrate for the G/T thymidine glycosylase (TDG) which corrects the G/T mispairs ^48-50.  In contrast, cytosine deamination ultimatively produces a CG →TA mutation in a two-step procedure. This CG →TA transition has been reported as being causally responsible for one-third of the single site mutations found in hereditary human diseases (reviewed by De Bont and van Larebeke, 2004) ⁵¹. It is of note that spontaneous base deamination predominates in single-stranded DNA compared to double-stranded DNA and is frequently provoked by transient single strandedness thoughout replication, transcription and recombination processes ⁵². However, exogenous stressors such as UV radiation ⁵³, sodium bisulfite ⁵⁴, nitrous acid ⁵⁵, and intercalating agents ⁵⁶ have also been identified to affect the amount of base deamination in the DNA. A growing body of evidence suggests that cytosine deamination induced by both, endogenous and exogenous stressors, plays a crucial role in mammalian evolution ^57-59.

 

Methylation of nitrogenous bases is the most common procedure of base alkylation, leading to the production of a wide diversity of modified bases such as N3-methyladenine, N3-methylcytosine, N7-methylguanine, N3-methylthymine and many more. Spontaneous DNA methylation can be induced, on the one hand, by S-adenosylmethionine (SAM), a regular methyl donor during physiological methylation reactions, leading to the formation of N3-methyladenine and N7-methylguanine ^{5, 51, 60}. On the other hand, several endogenous agents such as choline, betaine, and nitrosated bile salts have been shown to methylate DNA bases ⁶¹. Although methylation of DNA bases obviously represents an inferior structural modification, it may affect the chromatin structure as well as the regulation of gene expression as cytosine methylation in CpG islets is known to down-regulate gene expression. The available evidence indicates that dysregulated DNA methylation is a common feature of human cancers ^62-65. In this context, O6-methylguanine as well as O6-methylthymine and O4-ethylthymine have been found as being highly mutagenic by inducing the formation of GC→AT and TA→CG transitions, respectively ^66-69. Generation of methylated DNA bases represents a main prerequisite for the spontaneous occurrence of DNA lesions, if they cannot be fixed. Physiologically, methylated DNA bases are eliminated in either of two mechanisms: i) directly via O6-methylguanine DNA methyltransferase or though oxidation by an α-ketoglutarate-dependent dioxygenase AlkB homolog; ii) base excision repair (BER) with the participation of DNA glycosylases ^70-72.

 

Hydrolytic loss of nitrogenous bases in DNA is a further common variant of DNA damage ⁷³ which either manifests as spontaneous hydrolyzation of the N-glycosidic bond within the nucleotide or, enzymatically, as hydrolytic cleavage mediated by DNA glycosylases ⁷⁴. As demonstrated previously, DNA depyrimidination takes place much more slowly than depurination ^{75, 76}. The generation of abasic sites is strongly pH and temperature-dependent ⁷³ and, in turn, promotes the generation of single-strand breaks through a β-elimination reaction ^{77, 78} and/or mispairing ¹.

 

Oxidative DNA Damage

Oxidation of DNA is an ordinary procedure in physiological cell metabolism and frequently involved in regulation loops. Reactive oxygen species (ROS) play a predominant role in DNA oxidation as an excess of ROS is able to oxidize DNA at both, the desoxyribose moiety and the four nitrogenous bases. Notwithstanding this, physiological amounts of ROS function as signals in diverse cellular activities including pathogen defense, redox metabolism as well as stress and hormonal responses and development ^{79, 80}. ROS are typically generated during the mitochondrial respiration in aerobes and are by-products of catabolic oxidases, anabolic processes and the peroxisomal metabolism ⁸¹. The most important ROS comprise the superoxide radical (O₂^•−), hydrogen peroxide (H₂O₂), the hydroxyl radical (HO•), and singlet oxygen (¹O₂) ⁸². HO•, which is able to modify almost any class of macromolecules, accounts for the majority of DNA damages by attacking the C5 or  the C atom from the methyl group (CH₃) in pyrimidines, corresponding to the C8 in purines or the amino group of adenines ⁴⁵. HO• also attacks the carbohydrate moiety of nucleosides in close vicinity to the DNA bases ⁸³. Amongst the nitrogenous bases, guanine (G) is highly susceptible to ROS-mediated oxidation ⁸⁴. Oxidation of the C8 of the imidazole ring in deoxyguanosine (dG) causes the formation of 8-oxo-deoxyguanosine (8-oxo-dG) and its tautomer 8-hydroxy-deoxyguanosine (8-OH-dG), respectively ⁸⁵. As summarized by Giorgio et al. (2020), cellular dG is subjected to oxidation by a broad spectrum of reactive radicals (Figure 5) ⁸⁵. H₂O₂, generated by disproportionation of superoxide radicals that derive from molecular oxygen, represents the most prominent mediator in the formation of 8-oxo-dG as it facilitates the release of HO• through the Fenton reaction ⁸⁶. HO• can also be released from peroxynitrite (ONOO⁻) and peroxynitrous acid (HNO₃) ^{87, 88}. Notwithstanding the above, further reactive radicals such as singlet oxygen (¹O₂), peroxyl radical (HOO•), and carbonate radical anion (CO₃^•−) have also been found to contribute to the synthesis of 8-oxo-dG ^{85, 89, 90}. Apart from the formation of 8-oxo-dG, HO• has been described to mediate the opening of the imidazole ring in guanine and adenine leading to the formation of formamidopyrimidines (Figure 6) ¹. These adducts can be subjected to secondary modifications, yielding N5-substituted formamidopyrimidine (Fapy-dG) adducts ⁹¹. These Fapy-dG adducts are characterized by a pronounced mutagenic potential leading to base substitutions, among which G→T transitions are the most common ones ⁹².  N5-substituted Fapy-dG adducts have long been assumed as being eliminated by nucleotide excision repair (NER) ⁹³. Nevertheless, Vartanian and colleagues recently reported that this lesion is eliminated by base excision repair (BER) ⁹⁴.

 

Whereas oxidation of purine bases culminates in the synthesis of 8-oxo derivates, pyrimidine oxidation routinely leads to the formation of thymine glycol, uracil glycol, 5-hydroxycytosine and 5-hydroxyuracil ⁹⁵. In addition, endogenous lipid peroxidation mediated by HO• has been identified to form pro-mutagenic ethanobases (e.g., N6,1-ethenoadenine, N2,3-ethenoguanine) ^{96, 97}. Such DNA adducts have been reported to induce bp substitution mutations in prokaryotic and eukaryotic cells in vitro (for a review see Barbin, 2000) ⁹⁸. Lipid peroxidation also forms aldehyde species such as malondialdehyde and 4-hydroxynonenal that interact with adenine, guanine and cytosine to produce pro-mutagenic DNA adducts such as 8-hydroxy-propanodeoxyguanosine (HOPdG) ⁹⁹ and N^e-oxopropenyllysine ¹⁰⁰.

 

Besides their capacity to attack the four nitrogenous bases, ROS are able to oxidize DNA at the desoxyribose moiety, consequently leading to the formation of DNA single-strand breaks (SSB) and double-strand breaks (DSB). While SSB are generated by breakage of the phosphodiester bond between two adjacent deoxyribose moieties in the backbone of DNA, DSB result from the disruption of the phosphodiester bond of opposite strands within the same DNA molecule, the cleavage sites being in close vicinity to each other leading to the physical separation of the cracked ends. In contrast to the adjustment of oxidized bases via the BER, DNA strand breaks are corrected via single-strand break repair (SSBR) and the double-strand break repair (DSBR) response, respectively ^{101, 102}. If, however, left unrepaired, the persistence of DNA strand breaks may have detrimental consequences for the cell as it induces several chromosomal rearrangements affecting genome stability ¹.  Particularly in tumor cells, genetic instability is regarded as a hallmark of human cancers contributing to tumor initiation and progression ^{103, 104}.