**5. Mechanism of oxidative DNA base damage: The BER pathway**

As mentioned above, excess ROS may induce oxidative DNA damage, DNA strand breaks, base modifications and chromosomal aberrations [Marnett, 2000]. For repair of the DNA damage, human cells have five DNA repair systems: Direct reversal, mismatch repair, double-strand break repair, base excision repair (BER) and nucleotide excision repair (NER) [Wood et al., 2001]. DNA lesions resulting from oxidative stress, as well as single strand breaks, are repaired via the BER pathway in organisms ranging from E. coli to mammals (Figure 1) [Hazra et al., 2007, Krokan et al., 2003].

It is believed that DNA-BER is the major pathway for repairing deaminated bases and bases with oxidative damage generated by ROS and can be used to repair alkylated bases [Hazra et al., 2007, Hedge et al., 2008]. BER has 4 main steps (i.e. base removal, AP site incision, synthesis, and ligation) and involves 4 major classes of DNA repair enzymes: DNA glycosylases, APE, DNA polymerases, and DNA ligases.

The proteins involved in this reaction function in concert to remove a damaged DNA base and replace it with the correct base. A currently accepted model for the BER pathway reveals five distinct enzymatic steps for the repair of damaged bases.

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

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

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

As mentioned above, excess ROS may induce oxidative DNA damage, DNA strand breaks, base modifications and chromosomal aberrations [Marnett, 2000]. For repair of the DNA damage, human cells have five DNA repair systems: Direct reversal, mismatch repair, double-strand break repair, base excision repair (BER) and nucleotide excision repair (NER) [Wood et al., 2001]. DNA lesions resulting from oxidative stress, as well as single strand breaks, are repaired via the BER pathway in organisms ranging from E. coli to mammals

It is believed that DNA-BER is the major pathway for repairing deaminated bases and bases with oxidative damage generated by ROS and can be used to repair alkylated bases [Hazra et al., 2007, Hedge et al., 2008]. BER has 4 main steps (i.e. base removal, AP site incision, synthesis, and ligation) and involves 4 major classes of DNA repair enzymes: DNA

The proteins involved in this reaction function in concert to remove a damaged DNA base and replace it with the correct base. A currently accepted model for the BER pathway

identified in mammalian chromatin in vitro, but not in vivo [Dizdaroglu, 1998]

**5. Mechanism of oxidative DNA base damage: The BER pathway** 

(Figure 1) [Hazra et al., 2007, Krokan et al., 2003].

glycosylases, APE, DNA polymerases, and DNA ligases.

reveals five distinct enzymatic steps for the repair of damaged bases.

generating systems [Dizdaroglu, 1998].

sugar radical before cyclization.

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 or β, δ-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 components of which are coded by nuclear genes.

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 compatible with the downstream enzymatic reactions in BER.

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

Effect of Oxidative Stress on DNA Repairing Genes 59

The carcinogenicity of oxidative stress is primarily attributed to the genotoxicity of ROS in diverse cellular processes [ Ziech, 2011]. Oxidative mechanisms have been demonstrated to possess a potential role in the initiation, promotion, and malignant conversion (progression) stages of carcinogenesis [Cooke et al., 2003] . It has been suggested that some signaling system induces ROS that exhibit dual roles, cancer promoting and cancer suppresing, in tumorogenesis. ROS participate simultaneously in two signaling pathways that have inverse functions in tumorigenesis, Ras-Raf-MEK1/2-ERK1/2 signaling and the p38 mitogenactivated protein kinases (MAPK) pathway. Ras-Raf-MEK1/2-ERK1/2 signaling plays a role in oncogenesis, while the p38 MAPK pathway contributes to cancer suppression. The accumulation of intracellular ROS induced by oncogenic Ras is ERK-dependent during the

Increased intracellular levels of ROS, induced by the Ras-Raf-MEK-ERK signaling cascade, may mediate the activation of the p38 pathway and act as an intermediate signal between the MEK-ERK and MKK3/6-p38 pathways. On the one hand, the activation of p38 mitogenactivated protein kinase (MAPK) is a prerequisite for ROS-mediated functions such as apoptotic cell death in cancer cells. On the other hand, inhibiting or scavenging ROS may attenuate the activation of p38-dependent pathways. Human cancer cell lines with high ROS levels display enhanced tumorigenicity and impaired p38α activation by ROS [Pan et al.,

The effect of ROS by Ras may occur at the transcription level. GATA-6 is a component of the specific protein-DNA complexes at the nicotinamide adenine dinucleotide phosphate oxidase (Nox) 1 promoter, and is able to trans-activate the Nox1 promoter. GATA-6 is phosphorylated at serine residues by MEK-activated extracellular signal regulated kinase (ERK), which enhances GATA-6 DNA binding. The activity of the ROS-generating enzyme Nox1 is required for vascular endothelial growth factor (VEGF), a potent stimulator of tumor angiogenesis. Ras signaling enhances the transcription of Nox1 [Adachi et al., 2008]. A regulatory subunit, Rac, of the NADPH oxidase complex also involves the regulation of ROS [Heyworth et al., 1993, Kadara et al., 2008]. However, if extracellular signal-regulated kinase (ERK)-dependent phosphorylation of the transcription factor Sp1 and Sp1 binding to

Although some studies suggested that increased intracellular ROS elevate the activation of p38, these results have not been confirmed yet by the other researchers. It seems that further

Since the main purpose of this chapter is not to deal with the effect of ROS in tumorogenesis, we will explain the effect of ROS on DNA and DNA repair enzymes. ROS can cause direct

Major oxidative DNA damage products including those of 8-oxo-7, 8-dihydroadenine (8 oxoAde), 8-oxo-7, 8-dihydroguanine (8-oxoGua), 8-oxo-7, 8-dihydro-2′-deoxyguanosine (8-oxodG), and 5, 6-dihydroxy-5, 6-dihydrothymine as well as the ring-open lesions of 4, 6-diamino-5-formamido-pyrimidine and 2, 6-diamino-4-hydroxy-5-formamidopyrimidine [Kohen & Nyska, 2002]. Of these oxidative products, 8-oxoGua is known to be a biomarker of oxidative stres and its mutagenicity in mammalian cells demonstrates an additional potential as an intermediate marker of a disease endpoint (e.g. cancer). Elevated levels of such DNA lesions have been noted in many tumor types and are strongly implicated in the etiology of cancer [Valko et al., 2006]. Approximately 50%

a VEGF promoter is inhibited, this activity does not occur [Pan et al., 2009].

studies are needed to understand the mechanism of ROS in cancer.

oxidative DNA damage by increasing a cell's mutation.

**Cancer** 

2009].

activation of p38a [Pan et al., 2009)

for the redox activation of transcription factors (e.g. p53) spontaneously oxidized at cysteine residues in DNA binding domains to establish DNA binding activity [Jayaraman et al., 1997]. Deletion of APE in mice causes embryonic lethality [Jayaraman et al., 1997]. Cells from mice with APE haploinsufficiency respond poorly to oxidative stress [Meira et al., 2001].

The nick generated by the cleavage of the abasic site is filled in by either Pol or Pol / in the nucleus, and Pol in the mitochondria. Pol is the only DNA polymerase identified so far in vertebrate mitochondria and functions both as the replicative and the repair polymerase [Weissman et al., 2007, Kaguni, 2004]. Relevant to its role in BER, Pol has a dRP-lyase activity and can catalyze the 3'-end-processing necessary for short-patch BER [Longley et al., 1998].

The final step in the BER pathway is ligation of the nick with the correct nucleotide by DNA polymerases. An adenosine triphosphate-dependent DNA ligase (Ligase [Lig]I or III) completes the repair process and restores the integrity of the helix by sealing the nick. In the nucleus, two distinct DNA ligases participate in BER, ligase I, which has been implicated in long-patch BER, and ligase III, implicated in short-patch. The human ligase III gene (*LIG3*) also encodes for a mitochondrial variant, with a putative MTS generated by an alternative downstream translation initiation site [Lakshmipathy & Campbell, 1999, Lakshmipathy & Campbell, 2001]. The localization of ligase III protein to mitochondria suggests, then, that this enzyme may perform the ligation step in mtBER. Accordingly, reduction of ligase III expression using an antisense strategy resulted in an increase in breaks in mtDNA [Lakshmipathy & Campbell, 2001].
