**1. Introduction**

304 Selected Topics in DNA Repair

Yuan, Z. M., Utsugisawa, T., Ishiko, T. et al. (1998). Activation of protein kinase C delta

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by the c-Abl tyrosine kinase in response to ionizing radiation. Oncogene

The apurinic/apyrimidinic (AP) sites (called also abasic sites) are common lesion in genomic DNA, arising at a frequency of 10,000 to 50,000 lesions per mammalian cell per day (Lindahl, 1993). Unrepaired AP sites present mutagenic and cytotoxic consequences to the cell (Wilson; & Thompson, 1997). Most of the abasic sites are believed to result directly from spontaneous depurination, or indirectly from deamination of cytosine to uracil, which is then eliminated by uracil glycosylases. AP sites also result from hydrolysis of oxidized or alkylated bases by lesion-specific glycosylases during the early stage of base excision repair (BER) (McCullough et al., 1999). AP sites in isolated DNA are rather stable, but can be converted to single-strand breaks by alkali treatment, heating or nucleophilic attack at the aldehydic C1' group (Burrows & Muller, 1998). Intact abasic sites are noncoding lesions and *in vivo* can be stable enough to be mutagenic during DNA replication (Loeb & Preston, 1986). To protect genome integrity, eukaryotic organisms have robust enzyme activities, mainly APE1 in mammalian cells (Wilson & Barsky, 2001), that recognize abasic sites and cut the DNA backbone initiating the repair process. The continuous generation and repair of AP sites results in a steady-state levels of AP sites in mammalian cells in the range of approximately 1 site per 106 nucleotides (Atamna et al., 2000, Mohsin Ali et al., 2004). The number of AP sites can increase dramatically under stressful conditions such as X-ray or UV light irradiation or oxidative and alkylating agent exposure (Atamna et al., 2000). Considering the ubiquity of these lesions, it is reasonable to assume that wide range of cellular proteins can interact with abasic sites depending on the physiological state and stages of cell cycle.

The chapter is devoted to search of previously unrecognized proteins capable to interact with intact or cleaved AP sites. We mainly focused on proteins that form Schiff base upon this interaction. In most cases, these proteins are able to process AP sites although less efficiently than previously known counterparts. The biological role of these interactions in providing of backup pathways of DNA repair processes is also discussed.

### **2. Proteins that recognize AP sites**

In genomic DNA of higher eukaryotes AP sites irrespectively of their origin are thought to be repaired by base excision repair (Almeida & Sobol, 2007; Hegde et al., 2008).

New Players in Recognition of Intact and Cleaved AP Sites:

**2.2 Processing of AP sites** 

the BER process.

Implication in DNA Repair in Mammalian Cells 307

irradiation or ROS (Fan & Wilson, 2005). AP sites appeared via spontaneous base loss and

AP sites can be incised by three mechanistically different ways represented in Fig.1.

Fig. 1. Structures of intact and cleaved AP sites. DNA structures containing reactive sugar

In mammalian cells, the repair of AP sites is generally initiated through strand incision by APE1, the second enzyme of the canonical BER pathway (Demple et al., 1991). APE1 is thought to process of over 95% of AP sites in mammalian cells (Demple & Harrison, 1994; Wilson & Barsky, 2001). APE1 catalyzes hydrolysis of AP sites by a Mg2+-dependent mechanism, resulting in cleavage of the phosphodiester bond 5' to the AP site and generation of a single-strand break. This reaction produces a 3'-hydroxyl group and a 5' dRP group flanking the break. This way of AP site processing fits short-patch pathway of

Another way of AP site cleavage by the BER machinery involving the action of bi-functional DNA glycosylases is more complicated and requires the 'end cleaning' of termini prior to the further repair reactions may occur. An intermediate reaction step for these enzymes is the formation of a transient Schiff base between the amino group and the C1′ of deoxyribose for both base excision and subsequent DNA strand cleavage. In solution, AP sites exist predominantly as a mixture of ring-closed α- and β-hemiacetals with a minor amount of ring-opened aldehyde and aldehyde hydrate (<1%) (de los Santos et al., 2004). AP sites can be incised through -elimination using ε-NH2 of a lysine as the active site nucleophile (David & Williams, 1998; McCullough et al., 1999). Strand incision by -elimination forms a nick with phospho-α,β-unsaturated aldehyde (PUA) at the 3' margin and phosphate at the 5' margin (David & Williams, 1998; McCullough et al., 1999) (Fig. 1). While recently discovered mammalian DNA glycosylases, termed NEIL (Nei-like)-1, -2, catalyze the ,δ-elimination

moieties capable of Schiff base formation are marked by grey rectangular boxes.

arising from irradiation are also further processed by the BER machinery.

#### **2.1 Base excision repair (overview)**

Base excision repair (BER) is one of the major systems of DNA repair, mostly responsible for removing from DNA of non-bulky base lesions that appear in the genome with high frequency (Almeida & Sobol, 2007; Hegde et al., 2008; Schärer, 2003). The base excision repair pathway essentially removes and replaces nucleotides containing aberrant bases in DNA. Metabolically produced reactive nitrogen and oxygen species can modify the DNA bases due to oxidation, deamination and even alkylation at several positions in the base. BER is usually defined as DNA repair initiated by a lesion-specific DNA glycosylase and completed by either of two sub-pathways: short-patch BER; a mechanism whereby only 1 nucleotide is replaced or long-patch BER; a mechanism whereby 2–13 nucleotides are replaced (Frosina et al., 1996; Klungland & Lindahl, 1997).

The majority of BER is currently thought to occur via the short-patch pathway initiated by either a mono-functional or bi-functional DNA glycosylases. The short-patch BER pathway mediated by a mono-functional glycosylase involves removal of aberrant base by the lesionspecific DNA glycosylases, enzymes that hydrolyze cleavage of *N*-glycosylic bond between base and deoxyribose. This results in apurinic/apyrimidinic (also termed abasic) sites (AP sites), which repair is generally initiated through strand incision at its 5'-side by an AP endonuclease, leaving a nick flanked by a 3'-hydroxyl of an undamaged and a deoxyriboso-5'-phosphate (dRP), to which the damaged base was formerly linked. DNA polymerase then inserts a normal deoxyribonucleotide; however, the ligation step to restore intact DNA is blocked because of the dangling dRP moiety. The situation is resolved by a special enzymatic activity, excising dRP moiety (short-patch), or by continuing DNA synthesis with strand displacement, followed by removal of the displaced strand by flap endonuclease 1 (long-patch BER) (Frosina et al., 1996; Klungland & Lindahl, 1997).

In mammalian cells, the major dRP-removing enzyme is DNA polymerase β (Pol β), a multifunctional enzyme consisting of the 8-kDa amino-terminal domain with deoxyribose phosphate (dRP) lyase activity and the 31-kDa carboxy-terminal domain with nucleotidyl transferase activity (Allinson et al., 2001, Matsumoto & Kim, 1995, Piersen et al., 1996, Podlutsky et al., 2001). Thus, Pol β can mediate both steps in single nucleotide BER: insertion of deoxyribonucleotide and removal of dRP moiety preparing the strand for ligation.

Oxidized bases in DNA are manly produced by reactive oxygen species (ROS). ROS are generated as by-products of metabolic processes, primarily oxidative metabolism in the mitochondria (Dawson et al., 1993), and pathological conditions such as inflammation. ROS are also generated by ionizing radiation and some chemotherapeutic drugs (Almeida & Sobol, 2007; Hegde et al., 2008; Schärer, 2003). Oxidized bases are primarily removed by bifunctional DNA glycosylases that have an additional AP site cleavage activity. In addition to oxidation of DNA bases, ROS attack deoxyribose in DNA to generate strand breaks with nonligatable ends. The 3′ blocking groups include 3′ phosphate, 3′ phosphoglycolaldehyde, or 3′ phosphoglycolate (Breen et al., 1995). The 5′ terminus normally contains phosphate but after ROS reaction the nonligatable ends include 5′ OH and 5′ phosphodeoxyribose derivatives such as 2-deoxyribonolactone (Demple et al., 2002). Repair of single-strand breaks (SSBR) with blocked termini utilizes many of the same proteins as the BER process. The principal difference between SSBR and BER is the initiation step. Further both processes may occur at the single nucleotide level (i.e., short-patch repair) or as a long-patch of repair. BER is initiated via DNA glycosylase activity that results in the removal of a damaged base. SSBR is defined specifically for the repair of single-strand breaks in DNA generated by irradiation or ROS (Fan & Wilson, 2005). AP sites appeared via spontaneous base loss and arising from irradiation are also further processed by the BER machinery.
