**2.2 Processing of AP sites**

306 Selected Topics in DNA Repair

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

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

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

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

**2.1 Base excision repair (overview)** 

ligation.

replaced (Frosina et al., 1996; Klungland & Lindahl, 1997).

(long-patch BER) (Frosina et al., 1996; Klungland & Lindahl, 1997).

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

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 the BER process.

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

New Players in Recognition of Intact and Cleaved AP Sites:

cleaved AP site.

*cerevisiae* was reported in (Rieger et al., 2006).

Implication in DNA Repair in Mammalian Cells 309

Fig. 2. Workflow of identification of protein cross-linked to DNA containing intact or

isolation of cross-linked DNA-protein products; (3) the preparative cross-linking of extract proteins with such DNA; (4) the affinity purification of the resulted products; (5) the identification of the protein in the covalent adduct with DNA using mass-spectrometry methods; (6) the confirmation of the results using purified proteins and/or specific antibodies and the known functions/interactions of the identified protein; and (7) the study of the functional role of the revealed interactions between proteins and AP DNA. A proofof-principle identification of protein reactive towards AP sites in species from *E. coli* and *S.* 

It should be noted that used protocol includes two steps that increases the selectivity of the method. Affinity purification of cross-linked protein was carried out on commercially available affinity sorbents containing streptavidin, which binds the biotin residue. After adsorption, non-specifically bound proteins were removed by a series of washings. Additional separation of proteins was achieved by electrophoresis under denaturing conditions by the Laemmli method (Laemmli, 1970) followed by staining proteins. The target product was identified by the colocalization in the gel positions of the label in DNA and the stained protein. This approach indicates that only the protein cross-linked to DNA will be subjected to the further analysis. However, one cannot fully exclude the presence of

The identification was carried out for a number of proteins forming cross-links with AP sites in extracts of *E. coli* cells at logarithmic and stationary phases of cell growth (Rieger et al., 2006). It should be noted that authors used the previously developed non-enzymatic approach of the creation of AP sites in DNA based on the periodate oxidation of 2,3,5,6 tetrahydroxyhexyl phosphate precursor, which was introduced into an oligonucleotide

impurity proteins with the same electrophoretic mobility as the target product.

reaction with the N-terminal proline being used as the nucleophile (Hazra et al., 2002; Takao et al., 2002, Das et al., 2006). The ,δ-elimination reaction results in a one nucleotide gap flanked with phosphate groups at the 3'- and 5' margins. The products of bi-functional DNA glycosylase activity require the trimming of the 3'-ends to produce 3'-end hydroxyl groups that are indispensable for DNA polymerase activity.

Most of mammalian proteins known to form the Schiff base intermediate with the AP site appear to belong to the BER system. On the other hand, an interaction of AP DNA with proteins not formally involved in BER (for example, human ribosomal protein S3 and nucleoside diphosphate kinase – NM23-H2/NDP) has been well documented (Hegde et al., 2004; Postel et al., 2000). These two proteins are able to cleave AP sites. MutY, unlike other monofuctional DNA glycosylases, is able to interact with AP site via a Schiff base formation (Zharkov & Grollman, 1998) without the concomitant cleavage of AP sites. In this particular case formation of the Schiff base intermediate is considered as a mechanism for temporal protection of AP sites (Zharkov & Grollman, 1998).

Since AP sites in DNA appear to be promiscuous in their binding to many cellular factors and they are constantly generated in high frequency in genomic DNA, it may be important to sequester the AP sites immediately upon formation and further process them by the way that is most favorable for DNA integrity. Thus, interaction of cellular proteins with AP sites might be important for their repair/temporal protection from further degradation or might be involved in damage sensing/signaling making search of AP site reactive proteins a very important task.

#### **2.3 Search of new participants of recognition/processing of AP sites**

The very promising approach in search for unknown players in AP site recognition is based on a well-known propensity of deoxyribose in AP site, existing in equilibrium between cyclic furanose and acyclic aldehyde forms, to react with amine moieties in its vicinity. The Schiff base intermediate can be reduced by sodium borohydride (NaBH4) or related compounds, forming an irreversible complex between the enzyme and DNA (David & Williams, 1998; Piersen et al., 2000). This reaction is widely used to prove the β-elimination reaction mechanism for the enzymes capable of AP site cleavage (David & Williams, 1998; Piersen et al., 2000), although some proteins (e.g. MutY DNA glycosylase) can form a Schiff base with no further β-elimination (Zharkov & Grollman, 1998). Therefore, upon searching of new players in AP site recognition mediated by the Schiff base formation their ability to cleave AP has to be proved. Along with intact AP site, the product of its cleavage – 3' PUA and 5' dRP moieties (Fig. 1, grey rectangular boxes) are also able to form the Schiff base intermediate with primary amino groups of proteins that allows to use these DNA in search and identification of proteins.

#### **2.3.1 Identification of proteins reactive to AP sites**

For identification of protein cross-linked to AP DNA in cell extracts immunochemical and/or mass-spectrometry methods can be used. Identification of proteins reactive to AP sites by combination of cross-linking technique and mass-spectrometry is schematically represented in Fig. 2.

In general, search and identification of proteins reactive to AP sites include the following steps: (1) finding of cellular extract proteins forming covalent adducts with AP sites in DNA; (2) the design of AP-DNA probe containing a functional group providing the selective

reaction with the N-terminal proline being used as the nucleophile (Hazra et al., 2002; Takao et al., 2002, Das et al., 2006). The ,δ-elimination reaction results in a one nucleotide gap flanked with phosphate groups at the 3'- and 5' margins. The products of bi-functional DNA glycosylase activity require the trimming of the 3'-ends to produce 3'-end hydroxyl groups

Most of mammalian proteins known to form the Schiff base intermediate with the AP site appear to belong to the BER system. On the other hand, an interaction of AP DNA with proteins not formally involved in BER (for example, human ribosomal protein S3 and nucleoside diphosphate kinase – NM23-H2/NDP) has been well documented (Hegde et al., 2004; Postel et al., 2000). These two proteins are able to cleave AP sites. MutY, unlike other monofuctional DNA glycosylases, is able to interact with AP site via a Schiff base formation (Zharkov & Grollman, 1998) without the concomitant cleavage of AP sites. In this particular case formation of the Schiff base intermediate is considered as a mechanism for temporal

Since AP sites in DNA appear to be promiscuous in their binding to many cellular factors and they are constantly generated in high frequency in genomic DNA, it may be important to sequester the AP sites immediately upon formation and further process them by the way that is most favorable for DNA integrity. Thus, interaction of cellular proteins with AP sites might be important for their repair/temporal protection from further degradation or might be involved in damage sensing/signaling making search of AP site reactive proteins a very

The very promising approach in search for unknown players in AP site recognition is based on a well-known propensity of deoxyribose in AP site, existing in equilibrium between cyclic furanose and acyclic aldehyde forms, to react with amine moieties in its vicinity. The Schiff base intermediate can be reduced by sodium borohydride (NaBH4) or related compounds, forming an irreversible complex between the enzyme and DNA (David & Williams, 1998; Piersen et al., 2000). This reaction is widely used to prove the β-elimination reaction mechanism for the enzymes capable of AP site cleavage (David & Williams, 1998; Piersen et al., 2000), although some proteins (e.g. MutY DNA glycosylase) can form a Schiff base with no further β-elimination (Zharkov & Grollman, 1998). Therefore, upon searching of new players in AP site recognition mediated by the Schiff base formation their ability to cleave AP has to be proved. Along with intact AP site, the product of its cleavage – 3' PUA and 5' dRP moieties (Fig. 1, grey rectangular boxes) are also able to form the Schiff base intermediate with primary amino groups of proteins that allows to use these DNA in search

For identification of protein cross-linked to AP DNA in cell extracts immunochemical and/or mass-spectrometry methods can be used. Identification of proteins reactive to AP sites by combination of cross-linking technique and mass-spectrometry is schematically

In general, search and identification of proteins reactive to AP sites include the following steps: (1) finding of cellular extract proteins forming covalent adducts with AP sites in DNA; (2) the design of AP-DNA probe containing a functional group providing the selective

**2.3 Search of new participants of recognition/processing of AP sites** 

that are indispensable for DNA polymerase activity.

protection of AP sites (Zharkov & Grollman, 1998).

important task.

and identification of proteins.

represented in Fig. 2.

**2.3.1 Identification of proteins reactive to AP sites** 

Fig. 2. Workflow of identification of protein cross-linked to DNA containing intact or cleaved AP site.

isolation of cross-linked DNA-protein products; (3) the preparative cross-linking of extract proteins with such DNA; (4) the affinity purification of the resulted products; (5) the identification of the protein in the covalent adduct with DNA using mass-spectrometry methods; (6) the confirmation of the results using purified proteins and/or specific antibodies and the known functions/interactions of the identified protein; and (7) the study of the functional role of the revealed interactions between proteins and AP DNA. A proofof-principle identification of protein reactive towards AP sites in species from *E. coli* and *S. cerevisiae* was reported in (Rieger et al., 2006).

It should be noted that used protocol includes two steps that increases the selectivity of the method. Affinity purification of cross-linked protein was carried out on commercially available affinity sorbents containing streptavidin, which binds the biotin residue. After adsorption, non-specifically bound proteins were removed by a series of washings. Additional separation of proteins was achieved by electrophoresis under denaturing conditions by the Laemmli method (Laemmli, 1970) followed by staining proteins. The target product was identified by the colocalization in the gel positions of the label in DNA and the stained protein. This approach indicates that only the protein cross-linked to DNA will be subjected to the further analysis. However, one cannot fully exclude the presence of impurity proteins with the same electrophoretic mobility as the target product.

The identification was carried out for a number of proteins forming cross-links with AP sites in extracts of *E. coli* cells at logarithmic and stationary phases of cell growth (Rieger et al., 2006). It should be noted that authors used the previously developed non-enzymatic approach of the creation of AP sites in DNA based on the periodate oxidation of 2,3,5,6 tetrahydroxyhexyl phosphate precursor, which was introduced into an oligonucleotide

New Players in Recognition of Intact and Cleaved AP Sites:

with NaBH4 and analyzed.

nucleophiles is indispensable for efficient catalysis.

**2.3.3 AP site recognition by the 5'-dRP/AP lyase in PARP-1** 

of ~120-kDa that is reactive to AP site (Fig. 4A, lanes 1–4).

realized according to the scheme shown in Fig. 2.

Implication in DNA Repair in Mammalian Cells 311

Fig. 3. Interaction of Ku antigen with AP sites (From Ilina et al., 2008). (A) Cross-linking of proteins in HeLa cell extract (lane 1); without borohydride treatment (lane 2); AP DNA probe was replaced by the DNA duplex containing a THF residue (lane 3). (B) Specificity of the Ku80 antigen interaction with AP DNA. Cross-linking of the HeLa cell extract proteins to AP DNA was performed in the absence (lane 1) or presence of competitive DNA at different concentrations (lanes 2-7). The structures of competitive DNAs are shown at the top. Ratio of competitive DNA to DNA probe is shown at the bottom. (C) Influence of DNA-PK on the activity of APE1. (D) Estimation of the stability of Ku complex with AP DNA in HeLa cell extract. AP DNA was preincubated with HeLa cell extract for 15 min at 37º. Then excess of competitive DNA containing a THF residue was added, and the reaction mixture was further incubated at 37º for additional 4 hours. Aliquots at different times were reduced

appropriate AP lyase substrate and AP lyase activity test in the cell extracts deficient and proficient in Ku antigen unambiguously testify to the role of Ku antigen in processing of AP sites positioned near 5′ termini of DS breaks. Moreover, transfection of Ku deficient or proficient cells with variants of specifically designed substrate DNAs (with natural AP site or its AP lyase-resistant analog or without AP site) followed by PCR amplification of joining products and subsequent restriction analysis of amplicons fully confirmed the necessity of Ku antigen AP lyase activity for removal of near-end AP sites. Altogether the results obtained *in vitro* and *in vivo* testify to use of the 5′-dRP/AP lyase activity of Ku antigen for the excision of near-end abasic sites and explain higher radiosensitivity of mammalian cells deficient in Ku antigen, which is indispensable for classical NHEJ (Schulte-Uentrop et al., 2008). It is worthy of notice, that the same mechanism of AP site cleavage is used by two unrelated DNA repair systems and the suitable positioning of AP sites relative to active site

In further screening for proteins that are reactive to AP sites in addition to a linear DNA duplex with an AP site in the middle of the 32P-5'end-labeled strand, we used circular AP site-containing DNA to exclude interference by Ku80. Circular double-stranded DNA was synthesized, using single-stranded M13 DNA as template, in the presence of dUTP; then, AP sites were generated by uracil DNA glycosylase treatment (Khodyreva et al., 2010a; Khodyreva et al., 2010b). Unlike short duplex DNA with an AP site, that predominantly cross-linked Ku80 in HeLa cell extract (Ilina et al., 2008 and Fig. 4A, lane 5), the use of circular AP site-containing DNA allowed us to detect a novel protein with molecular mass

To identify the cross-linked protein large-scale cross-linking with the bovine testis nuclear extract (BTNE) and a biotin-containing linear AP DNA was performed. Identification was

during the standard phosphoroamidite oligonucleotide synthesis. The periodate oxidation allows one to obtain AP sites in an almost quantitative yield. This method, in the authors' opinion, has several advantages over the more commonly used method that is based on the removal of uracil residues using uracil DNA glycosylase.

The procaryotic proteins AroF, DnaK, MutM, PolA, TnaA, TufA, and UvrA from *E. coli*  and eucaryotic ARC1 and Ygl245wp from yeast were identified (Rieger et al., 2006). Protein Ygl245wp with an unknown but essential for cell viability function was prepared in the individual state after its coding sequence was cloned, and the recombinant plasmid was used for the production of the protein in *E. coli* cells followed by its isolation. The obtained protein was shown to bind to AP DNA forming Schiff bases; however, the biological significance of this interaction has not been established. DNA polymerase I, like other DNA polymerases of the A-family, has a weak AP lyase activity and dRP lyase activity (Pinz & Bogenhagen, 2000). However the biological significance of these activities *in vivo* has not been established. The ability of UvrA, the key component of the nucleotide excision repair (NER) in bacteria, to interact with AP sites in the UvrABC complex indicates the possible role of NER as a back-up pathway of AP site repair in bacteria (Showden et al., 1990).

#### **2.3.2 Identification of Ku80 subunit of human Ku antigen as a protein reactive to AP sites**

Using the same technique we screened mammalian cell extracts for proteins capable of binding AP site-containing DNA. In HeLa cell extract, a 32-bp DNA duplex with an AP site in the middle of the DNA chain was shown to cross-link predominantly to a protein forming a product with an apparent molecular mass of 95 kDa (Fig. 3) (Ilina et al., 2008).

The subsequent analysis of whole cell extracts of human lung fibroblasts, K-562, and MCF-7 cells revealed the products with the same electrophoretic mobility. The preferential crosslinking of this protein reflected extraordinary specificity in light of the multitude of proteins in the cell. If the AP site in DNA is replaced by its analog (the THF residue) or the treatment with NaBH4 is omitted, DNA–protein cross-linked products are not registered (Fig. 3).

In spite of efficient cross-linking of the above mentioned AP DNA to Ku80 polypeptide (as a part of DNA-PK) no cleavage of AP sites was observed (Ilina et al., 2008). Instead Ku80 formed with AP site a long-living Schiff base intermediate without the concomitant AP site cleavage just as was observed for monofunctional DNA glycosylase MutY and considered as a mechanism for temporal protection of AP sites (Zharkov & Grollman, 1998). AP lyase and 5'-dRP activities are distinctive features of the BER process (Almeida & Sobol, 2007; Hegde et al., 2008). The BER 5'-dRP/AP lyases usually function beyond the DS-breaks. But abasic sites associated with double-strand breaks can be generated by ionizing radiation, by treatment with radiomimetic drugs or as a result of attempted BER of complex damages (Yang et al., 2004). These specifically positioned lesions must be removed prior to or in the course of DS break repair. Ku antigen – DS-end binding protein of NHEJ – has been recently shown to act as a 5'-dRP/AP lyase near double-strand breaks (Roberts et al., 2010).

At DSB ends Ku is approximately tenfold more active than Pol β (Roberts et al., 2010), the most known mammalian 5'-dRP lyase (Matsumoto & Kim, 1995). At the same time, Ku was inefficient as AP lyase at AP sites situated at a distance longer than one helix turn from DS breaks just as was reported in (Ilina et al., 2008). NaBH4-dependent cross-linking of

during the standard phosphoroamidite oligonucleotide synthesis. The periodate oxidation allows one to obtain AP sites in an almost quantitative yield. This method, in the authors' opinion, has several advantages over the more commonly used method that is based on the

The procaryotic proteins AroF, DnaK, MutM, PolA, TnaA, TufA, and UvrA from *E. coli*  and eucaryotic ARC1 and Ygl245wp from yeast were identified (Rieger et al., 2006). Protein Ygl245wp with an unknown but essential for cell viability function was prepared in the individual state after its coding sequence was cloned, and the recombinant plasmid was used for the production of the protein in *E. coli* cells followed by its isolation. The obtained protein was shown to bind to AP DNA forming Schiff bases; however, the biological significance of this interaction has not been established. DNA polymerase I, like other DNA polymerases of the A-family, has a weak AP lyase activity and dRP lyase activity (Pinz & Bogenhagen, 2000). However the biological significance of these activities *in vivo* has not been established. The ability of UvrA, the key component of the nucleotide excision repair (NER) in bacteria, to interact with AP sites in the UvrABC complex indicates the possible role of NER as a back-up pathway of AP site repair in bacteria

**2.3.2 Identification of Ku80 subunit of human Ku antigen as a protein reactive to AP** 

a product with an apparent molecular mass of 95 kDa (Fig. 3) (Ilina et al., 2008).

Using the same technique we screened mammalian cell extracts for proteins capable of binding AP site-containing DNA. In HeLa cell extract, a 32-bp DNA duplex with an AP site in the middle of the DNA chain was shown to cross-link predominantly to a protein forming

The subsequent analysis of whole cell extracts of human lung fibroblasts, K-562, and MCF-7 cells revealed the products with the same electrophoretic mobility. The preferential crosslinking of this protein reflected extraordinary specificity in light of the multitude of proteins in the cell. If the AP site in DNA is replaced by its analog (the THF residue) or the treatment with NaBH4 is omitted, DNA–protein cross-linked products are not registered (Fig. 3). In spite of efficient cross-linking of the above mentioned AP DNA to Ku80 polypeptide (as a part of DNA-PK) no cleavage of AP sites was observed (Ilina et al., 2008). Instead Ku80 formed with AP site a long-living Schiff base intermediate without the concomitant AP site cleavage just as was observed for monofunctional DNA glycosylase MutY and considered as a mechanism for temporal protection of AP sites (Zharkov & Grollman, 1998). AP lyase and 5'-dRP activities are distinctive features of the BER process (Almeida & Sobol, 2007; Hegde et al., 2008). The BER 5'-dRP/AP lyases usually function beyond the DS-breaks. But abasic sites associated with double-strand breaks can be generated by ionizing radiation, by treatment with radiomimetic drugs or as a result of attempted BER of complex damages (Yang et al., 2004). These specifically positioned lesions must be removed prior to or in the course of DS break repair. Ku antigen – DS-end binding protein of NHEJ – has been recently shown to act as a 5'-dRP/AP lyase near double-strand breaks

At DSB ends Ku is approximately tenfold more active than Pol β (Roberts et al., 2010), the most known mammalian 5'-dRP lyase (Matsumoto & Kim, 1995). At the same time, Ku was inefficient as AP lyase at AP sites situated at a distance longer than one helix turn from DS breaks just as was reported in (Ilina et al., 2008). NaBH4-dependent cross-linking of

removal of uracil residues using uracil DNA glycosylase.

(Showden et al., 1990).

(Roberts et al., 2010).

**sites** 

Fig. 3. Interaction of Ku antigen with AP sites (From Ilina et al., 2008). (A) Cross-linking of proteins in HeLa cell extract (lane 1); without borohydride treatment (lane 2); AP DNA probe was replaced by the DNA duplex containing a THF residue (lane 3). (B) Specificity of the Ku80 antigen interaction with AP DNA. Cross-linking of the HeLa cell extract proteins to AP DNA was performed in the absence (lane 1) or presence of competitive DNA at different concentrations (lanes 2-7). The structures of competitive DNAs are shown at the top. Ratio of competitive DNA to DNA probe is shown at the bottom. (C) Influence of DNA-PK on the activity of APE1. (D) Estimation of the stability of Ku complex with AP DNA in HeLa cell extract. AP DNA was preincubated with HeLa cell extract for 15 min at 37º. Then excess of competitive DNA containing a THF residue was added, and the reaction mixture was further incubated at 37º for additional 4 hours. Aliquots at different times were reduced with NaBH4 and analyzed.

appropriate AP lyase substrate and AP lyase activity test in the cell extracts deficient and proficient in Ku antigen unambiguously testify to the role of Ku antigen in processing of AP sites positioned near 5′ termini of DS breaks. Moreover, transfection of Ku deficient or proficient cells with variants of specifically designed substrate DNAs (with natural AP site or its AP lyase-resistant analog or without AP site) followed by PCR amplification of joining products and subsequent restriction analysis of amplicons fully confirmed the necessity of Ku antigen AP lyase activity for removal of near-end AP sites. Altogether the results obtained *in vitro* and *in vivo* testify to use of the 5′-dRP/AP lyase activity of Ku antigen for the excision of near-end abasic sites and explain higher radiosensitivity of mammalian cells deficient in Ku antigen, which is indispensable for classical NHEJ (Schulte-Uentrop et al., 2008). It is worthy of notice, that the same mechanism of AP site cleavage is used by two unrelated DNA repair systems and the suitable positioning of AP sites relative to active site nucleophiles is indispensable for efficient catalysis.
