**4. ROS-mediated DNA damage**

Although DNA is a stable and well-protected molecule, ROS can interact with it and cause several types of damage: modification of DNA bases, single- and double-DNA breaks, loss of purines (apurinic sites), damage to the deoxyribose sugar, DNA-protein cross-linkage, and damage to the DNA repair system (Figure 1) [Kohen & Nyska, 2002].

Of the ROS, the highly reactive •OH reacts with DNA by addition to double bonds of DNA bases and by abstraction of an H atom from the methyl group of thymine and each of the C-H bonds of 2'-deoxyribose. Addition of •OH to the C5-C6 double bond of pyrimidines leads to C5-OH and C6-OH adduct radicals of cytosine and thymine and H atom abstraction from thymine results in the allyl radical. Adduct radicals differ in terms of their redox properties, C5-OH- and C6-OH-adduct radicals of pyrimidines possess reducing and oxidising properties, respectively [Sonntag, 1987, Steenken, 1987, Teoule, 1987, Cooke et al., 2003].

Pyrimidine OH-adduct radicals and the allyl radical are oxidised or reduced depending on their redox environment, redox properties and reaction partners, yielding a variety of products [Dizdaroglu, 1992, Evans et al., 2004]. Product types and yields depend on absence and presence of O2 and on other conditions [Cooke et al., 2003, Breen & Murphy, 1995]. In the absence of O2, the oxidation of C5-OH adduct radicals, followed by addition of OH\_ (or addition of water followed by deprotonation), leads to cytosine glycol and thymine glycol. The oxidation of the allyl radical of thymine yields 5-hydroxymethyluracil (5-OHMeUra) when molecular O2 adds to C5-OH-adduct radicals at diffusion-controlled rates generating C5-OH-6-peroxyl radicals, which subsequently eliminate O2•− followed by reaction with water (addition of OH−) to yield thymine and cytosine glycols [Evans et al., 2004, Sonntag, 1987, Teoule, 1987].

Addition of O2 to the allyl radical leads to 5-OHMeUra and 5-formyluracil (5-FoUra). Pyrimidine peroxyl radicals are also reduced and then protonated to give hydroxyhydroperoxides [Teoule, 1987], which decompose and yield thymine glycol, 5- OHMeUra, 5-FoUra, and 5-hydroxy-5-methylhydantoin [Wagner et al., 1994, Dizdaroglu et al., 1993a].

Cytosine products may deaminate and dehydrate. Cytosine glycol deaminates to give uracil glycol, 5-hydroxycytosine (5-OH-Cyt), and 5-hydroxyuracil (5-OH-Ura) that are unique in that they can deaminate and dehydrate. However, there is evidence that these four compounds (cytosine glycol, uracil glycol, 5-OH-Cyt, and 5-OH-Ura) may simultaneously be existed damaged DNA [Wagner et al., 1994, Dizdaroglu et al., 1993a]. In the absence of O2, C5-OH adduct radicals may be reduced, followed by protonation to give rise to 5-hydroxy-6 hydropyrimidines. 5-Hydroxy-6-hydrocytosine (5-OH-6-HCyt) readily deaminates into 5 hydroxy-6-hydrouracil (5-OH-6-HUra). Similarly, C6-OH adduct radicals of pyrimidines are reduced yielding 6-hydroxy-5-hydropyrimidines. Formation of these products are inhibited in the presence of O2, because O2 reacts with their precursors C5-OH- and C6-OH-adduct radicals at diffusion-controlled rates to yield peroxyl radicals and then other products.

Further reactions of C5-OH-6-peroxyl and C6-OH-5-peroxyl radicals of cytosine result in formation of 4-amino-5-hydroxy-2, 6(1H, 5H)-pyrimidinedione and 4-amino-6-hydroxy-2, 5(1H, 6H)-pyrimidinedione, respectively. The former deaminates to give dialuric acid, which is readily oxidised to yield alloxan [Dizdaroglu et al., 1993a, Behrend et al., 1989, Dizdaroglu 1993b]. The presence of alloxan in damaged DNA has been shown by its release from DNA by *E. coli* Nth [Dizdaroglu et al., 1993a].

Although DNA is a stable and well-protected molecule, ROS can interact with it and cause several types of damage: modification of DNA bases, single- and double-DNA breaks, loss of purines (apurinic sites), damage to the deoxyribose sugar, DNA-protein cross-linkage,

Of the ROS, the highly reactive •OH reacts with DNA by addition to double bonds of DNA bases and by abstraction of an H atom from the methyl group of thymine and each of the C-H bonds of 2'-deoxyribose. Addition of •OH to the C5-C6 double bond of pyrimidines leads to C5-OH and C6-OH adduct radicals of cytosine and thymine and H atom abstraction from thymine results in the allyl radical. Adduct radicals differ in terms of their redox properties, C5-OH- and C6-OH-adduct radicals of pyrimidines possess reducing and oxidising properties, respectively [Sonntag, 1987, Steenken, 1987, Teoule, 1987, Cooke et al., 2003]. Pyrimidine OH-adduct radicals and the allyl radical are oxidised or reduced depending on their redox environment, redox properties and reaction partners, yielding a variety of products [Dizdaroglu, 1992, Evans et al., 2004]. Product types and yields depend on absence and presence of O2 and on other conditions [Cooke et al., 2003, Breen & Murphy, 1995]. In the absence of O2, the oxidation of C5-OH adduct radicals, followed by addition of OH\_ (or addition of water followed by deprotonation), leads to cytosine glycol and thymine glycol. The oxidation of the allyl radical of thymine yields 5-hydroxymethyluracil (5-OHMeUra) when molecular O2 adds to C5-OH-adduct radicals at diffusion-controlled rates generating C5-OH-6-peroxyl radicals, which subsequently eliminate O2•− followed by reaction with water (addition of OH−) to yield thymine and cytosine glycols [Evans et al., 2004, Sonntag,

Addition of O2 to the allyl radical leads to 5-OHMeUra and 5-formyluracil (5-FoUra). Pyrimidine peroxyl radicals are also reduced and then protonated to give hydroxyhydroperoxides [Teoule, 1987], which decompose and yield thymine glycol, 5- OHMeUra, 5-FoUra, and 5-hydroxy-5-methylhydantoin [Wagner et al., 1994, Dizdaroglu et

Cytosine products may deaminate and dehydrate. Cytosine glycol deaminates to give uracil glycol, 5-hydroxycytosine (5-OH-Cyt), and 5-hydroxyuracil (5-OH-Ura) that are unique in that they can deaminate and dehydrate. However, there is evidence that these four compounds (cytosine glycol, uracil glycol, 5-OH-Cyt, and 5-OH-Ura) may simultaneously be existed damaged DNA [Wagner et al., 1994, Dizdaroglu et al., 1993a]. In the absence of O2, C5-OH adduct radicals may be reduced, followed by protonation to give rise to 5-hydroxy-6 hydropyrimidines. 5-Hydroxy-6-hydrocytosine (5-OH-6-HCyt) readily deaminates into 5 hydroxy-6-hydrouracil (5-OH-6-HUra). Similarly, C6-OH adduct radicals of pyrimidines are reduced yielding 6-hydroxy-5-hydropyrimidines. Formation of these products are inhibited in the presence of O2, because O2 reacts with their precursors C5-OH- and C6-OH-adduct

radicals at diffusion-controlled rates to yield peroxyl radicals and then other products.

from DNA by *E. coli* Nth [Dizdaroglu et al., 1993a].

Further reactions of C5-OH-6-peroxyl and C6-OH-5-peroxyl radicals of cytosine result in formation of 4-amino-5-hydroxy-2, 6(1H, 5H)-pyrimidinedione and 4-amino-6-hydroxy-2, 5(1H, 6H)-pyrimidinedione, respectively. The former deaminates to give dialuric acid, which is readily oxidised to yield alloxan [Dizdaroglu et al., 1993a, Behrend et al., 1989, Dizdaroglu 1993b]. The presence of alloxan in damaged DNA has been shown by its release

and damage to the DNA repair system (Figure 1) [Kohen & Nyska, 2002].

**4. ROS-mediated DNA damage** 

1987, Teoule, 1987].

al., 1993a].

Decarboxylation of alloxan yields 5-hydroxyhydantoin (5-OH-Hyd) upon acidic treatment. Isodialuric acid is formed by deamination of 4-amino-6-hydroxy-2, 5(1H, 6H) pyrimidinedione. However, these two compounds may simultaneously exist in DNA as evidenced from the detection of their enol forms (5, 6-dihydroxycytosine (5, 6-diOH-Cyt) and 5, 6-dihydroxyuracil (5, 6-diOH-Ura), respectively) [11, 13]. 5-OH-6-hydroperoxide of cytosine undergoes intramolecular cyclisation to yield *trans*-1-carbamoyl-2-oxo-4, 5 dihydroxyimidazolidine as a major product in cytosine [Kohen & Nyska, 2002] However, this compound is formed as a minor product in DNA [Dizdaroglu et al., 1993a, Behrend et al., 1989, Dizdaroglu 1993b].

Hydroxyl adduct radicals of guanine are formed by addition of •OH to the C4-, C5- and C8 positions of guanine, generating C4-OH-, C5-OH- and C8-OH-adduct radicals [O'Neill, 1983, Steenken, 1989, Candeisas & Steenken, 2000]. The 6-substituted purines such as adenine undergo analogous reactions, yielding at least two OH adducts, are formed: C4-OH and C8-OH adduct radicals [Steenken, 1989, Candeisas & Steenken, 2000]. C4-OH and C5- OH adduct radicals of purines differ in their redox properties, with C4-OH-adduct radicals being oxidising, and C5-OH- and C8-OH-adduct radicals being primarily reducing.

On the other hand, different mesomeric structures of these radicals may be oxidizing or reducing, a phenomenon called "redox ambivalence" [Vieira & Steenken, 1990]. Dehydration of C4-OH- and C5-OH-adduct radicals of purines reconstitutes the purine by first yielding a purine (–H)• radical, followed by reduction and protonation [Melvin et al., 1996]. The C4-OH-adduct radical of guanine also eliminates OH− to give rise to the guanine radical cation (guanine•+), which may deprotonate depending on pH to give guanine(–H)• [Moreno & Pryor, 1992]. Furthermore, on the basis of product analysis, the radical cation does not hydrate to lead to the C8-OH adduct radical and then to 8-hydroxyguanine by oxidation. However, it may react with 2'-deoxyribose in DNA by H abstraction, leading to DNA strand breaks [O'Neill & Chapman, 1985].

This diversity has been explained by the notion that the hydration of guanine•+ in ds-DNA may be much faster than that of monomeric guanine•+ [Candeias & Steenken, 2000]. O<sup>2</sup> readily reacts with guanine(–H)•; however, its reaction with the C4-OH-adduct radical of guanine is rather slow [Candeias & Steenken, 2000]. The reaction of guanine(-H)• with O<sup>2</sup> leads to imidazolone and oxazolone derivatives [Cadet et al., 1991]. However, this suggestion has not been confirmed by pulse radiolysis data and an alternative mechanism has been proposed [Melvin et al., 1996]. The C4-OH-adduct radical of adenine readily reacts with O2, however, final products of this reaction are not known [O'Neill & Chapman, 1985]. C8-OH adduct radicals of purines may be oxidized by oxidants including O2. In contrast to C4-OH adduct radicals, their reaction with O2 is diffusion controlled [Vieira & Steenken, 1990].

8-Hydroxypurines are formed in DNA by the one-electron oxidation of C8-OH-adduct radicals [Bielski & Cabelli, 1995, Halliwell & Gutteridge, 1999] This reaction competes with the unimolecular opening of the imidazole ring by scission of the C8–N9 bond [Steenken, 1989, Candeias & Steenken, 2000]. The one-electron reduction of the ring-opened radical leads to 2, 6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) from guanine and 4, 6 diamino-5-formamidopyrimidine (Fapy- Ade) from adenine [O'Neill, 1983, Breen & Murphy, 1995]. The C8-OH adduct radicals may also be reduced without ring opening to give rise to 7-hydro-8-hydroxypurines, which, as hemiorthoamides, are converted into formamidopyrimidines. However, 8-Hydroxypurines and formamidopyrimidines are unique in that they are formed in DNA both in the absence and presence of O2, although O2 increases the yields of 8-hydroxypurines. Moreover, other experimental conditions highly

Effect of Oxidative Stress on DNA Repairing Genes 57

Glycosylases at the initial step of BER. DNA glycosylases make BER possible – glycosylases recognize specific damaged bases and excise them from the genome, effectively initiating both longpatch and short-patch BER. To date, 11 different mammalian glycosylases have been characterized. The primary function of most DNA glycosylases is to recognize their substrate (the damaged base) and catalyze the cleavage of an N-glycosidic bond, releasing a free base and creating an abasic site [Lindahl, 1974]. In addition to catalyzing the cleavage of N-glycosydic bonds, some glycosylases are bifunctional having an additional AP lyase activity [O'Connor & Laval, 1989]. The uracil-DNA glycosylase (UNG) was the first DNA glycosylase identified and cloned [Lindahl, 1974]. MPG, N-methylpurine-DNAglycosylase, and 8-oxoguanine-DNA glycosylase (OGG1)represent some extensively studied DNA glycosylases [Sedgwick et al., 2007, Kavli et al., 2007, Klungland & Bjelland, 2007, Robertson et al., 2009]. After recognition of the damaged base by the appropriate DNA glycosylase, this glycosylase catalyzes the cleavage of an N-glycosidic bond, effectively removing the damaged base and creating an apurinic or apyrimidinic site (AP site). The DNA backbone is cleaved by either a DNA AP endonuclease or a DNA AP lyase – an activity present in some glycosylases. AP endonuclease activity creates a single-stranded DNA nick 5' to the AP site, contrasting with the nick being created 3' to the AP site as a result of activity (β-elimination

Repair can then proceed through one of two subpathways: short or long-patch BER. The short-patch BER involves the incorporation of a single nucleotide into the gap by DNA polymerase followed by strand ligation by DNA ligase. The long-patch BER involves incorporation of several nucleotides, typically two to seven, followed by cleavage of the resulting 5' flap and ligation. Mitochondria possess independent BER machinery, the

Most DNA glycosylases have broad substrate specificities but can have preferences for either purines or pyrimidines. The major bifunctional glycosylase for purines is 8 oxoguanine DNA glycosylase 1, which removes OHdG and 8-oxoG. Most oxidized pyrimidines are removed by endonuclease III-like protein, uracil DNA glycosylase (UNG), and Nei-like DNA glycosylase (NEIL-1 and NEIL-2). In human cells, there are at least 2 isoforms of OGG1 (α and β) that arise from alternative splicing of products of the OGG1 gene [Boeiteux & Radicella, 2000]. α-OGG1 is a 345-amino acid (~39 kd) protein that localizes to the nucleus and mitochondria, whereas β-OGG1 is a 424-amino acid protein (47 kd) that seems to localize exclusively to mitochondria [Boeiteux & Radicella, 2000]. Because of the absence of an αO helix domain, β-OGG1 seems to lack glycosylase activity [Hashiguchi et al., 2004]. Endonuclease III-like protein 1 is a 312-amino acid (~34 kd) protein that localizes to the nucleus. OGG1 and endonuclease III-like protein require duplex DNA for efficient repair. After the glycosylase reaction, the newly created nick is processed by the Apurinic/apyrimidinic endonuclease (APE), creating a single-nucleotide gap in the DNA. Importantly, the gap created contains a 3'- hydroxyl and a 5'-phosphate, substrates

APE cleaves intact AP sites by making a single nick 5′ to the AP site to create 3′-OH and 5′ deoxyribose phosphate termini that are removed by the activity of 3′-OH and 5′-deoxyribose phosphatase proteins such as DNA Polβ. The 3-phosphate removal activity of APE is, however, approximately 70-fold lower than the AP endonuclease activity of APE [Winters et al., 1994], suggesting that other enzymes are important for the processing of damaged 3′ ends [Takahashi et al., 2007] APE is a 317-amino acid (~37 kd) multifunctional protein localized to the nucleus [Demple et al., 1991]. In addition to its role in BER, APE is required

or β, δ-elimination).

components of which are coded by nuclear genes.

compatible with the downstream enzymatic reactions in BER.

affect the yields of these compounds, such as the presence of reducing or oxidizing agents [Dizdaroglu, 1992, Breen & Murphy, 1995]. In the case of adenine, the •OH attack at the C2 position has also been proposed to take place and yield 2-hydroxyadenine (2-OH-Ade) when oxidation of the thus-formed C2-OH-adduct radical [Nackerdien et al., 1991]. Reactions of pyrimidines and purines result in multiple products in DNA. Most of these modified bases were identified in DNA in vitro and in vivo upon exposure to free radical generating systems [Dizdaroglu, 1998].

Reactions of •OH with the sugar moiety of DNA by H abstraction give rise to sugar modifications. Some sugar products are released from DNA as free modified sugars, whereas others remain within DNA or constitute end groups of broken DNA strands. A unique reaction of the C5'-centered sugar radical is the addition to the C8- position of the purine ring of the same nucleoside. This intramolecular cyclisation followed by oxidation yields 8, 5'-cyclopurine-2'-deoxynucleosides [Dizdaroglu, 1986, Dirksen et al., 1988]. Both 5'R- and 5'S-diastereomers of 8, 5'-cyclo-2'-deoxyguanosine (cyclo-dG) and 8, 5'-cyclo-2' deoxyadenosine (cyclo-dA) are generated in DNA[Dizdaroglu, 1986, Dirksen et al., 1988]. (5'R)-and (5'S)- cyclo-dGs were also identified in human cells exposed to ionizing radiation [Dizdaroglu, 1987]. These tandem lesions represent a concomitant damage to both the base and sugar moieties of DNA. O2 inhibits their formation by reacting with the C5' centered sugar radical before cyclization.

Another reaction of base radicals is the addition to an aromatic amino acid of proteins or combination with an amino acid radical, leading to DNA–protein cross-linking [Dizdaroglu, 1988]. Covalent DNA–protein cross-links are formed in cells in vivo or in chromatin in vitro by exposure to free radical-generating systems, e.g., ionising radiation. The formation of a thymine–tyrosine cross-link has been observed in mammalian chromatin in vitro and in living cells upon exposure to ionising radiation, H2O2, metal ions and carcinogenic compounds [Dizdaroglu et al. 1989, Nackerdien et al., 1991, Olinski et al., 1992, Altman et al., 1995]. The formation mechanism of DNA–protein cross-link has been proposed to involve the addition of the allyl radical of thymine in DNA to tyrosine in a protein, followed by oxidation. Chemical structures of other DNA base-amino acid cross-links have been identified in mammalian chromatin in vitro, but not in vivo [Dizdaroglu, 1998]
