**2.3.5 Identification of HMGB1 as cofactor of the BER process**

To identify proteins that have dRP lyase activity or influence removal of the dRP from BER intermediates in the absence of Pol β, we used Pol β null mouse embryonic fibroblast (MEF) cell extract for sodium borohydride driven cross-linking of the Schiff base dRP lyase intermediate protein-DNA complex (Prasad et al., 2007). The strong labeling of a single species in the Pol β null cell extracts corresponding to an unknown protein-DNA complex of 37 kDa was observed (Fig. 6A).

The preferential cross-linking of this protein reflected extraordinary specificity in light of the multitude of proteins in the cell. Taking into account the molecular masses of NEIL1 and NEIL2 (43.5 kDa and 38.2 kDa, respectively) the product could not be related to glycosylases. It should be noted that an apparent molecular mass of a covalent adduct protein-nucleic acid estimated by electrophoretic mobility is approximately equal to the sum of molecular masses of protein and attached nucleic acid.

For identification of protein we applied the approach schematically depicted in Fig. 2. The DNA probe contained a 32P-labeled dRP moiety in a single-stranded break and a 3′-biotin tag to facilitate isolation of cross-linked protein-DNA complexes. Eleven of ions observed in MALDI MS spectrum corresponded to peptides of HMGB1. The (M + H)+ ion of m/z 1520.84 was selected automatically during the data dependent acquisition for MS/MS analysis. The values from both the peptide masses and the MS/MS fragment ion masses were used in a database search. The protein was identified as HMGB1 with a Mowse-based score of 102, 32% sequence coverage and a protein score confidence interval of 99.995%. Among the observed ions the ion of m/z 1520.84 corresponds to amino acid residues 113– 127 of the mouse HMGB1 and is a 'signature' that distinguishes HMGB1 from the closely related protein, HMGB2 (Bustin & Reeves, 1996). HMGB1 and HMGB2 are nuclear nonhistone DNA-binding proteins that belong to the high-mobility group box family of proteins (Bustin & Reeves, 1996). HMGB1 has an architectural role in the assembly of nucleoprotein complexes and is highly conserved across species (Bustin & Reeves, 1996; Tang et al., 2010 ;Liu et al., 2010; Stros, 2010). HMGB1 binds to DNA in the minor groove without sequence specificity and has the ability to transiently introduce bends or kinks into linear DNA (Liu et al., 2010; Stros, 2010). The intrinsic ability of HMGB1 to alter DNA structures allows it to participate in many biological processes including regulation of

The experiments with individual enzymes suggest that NEIL1 and NEIL2 possess dRP lyase activities and could substitute for Pol β in removing dRP moiety in the BER process. To analyze the proficiency of NEIL1 and NEIL2 dRPase in a multienzyme BER process, we have reconstituted the base-excision, AP site-incision, gap-filling and dRP-excision stages of BER using mammalian enzymes (UNG, OGG1, APE1, Pol β (wild type and K35A/K68A/K72A mutant deficient in dRP lyase activity) and NEIL1 or NEIL2. Both NEIL1 and NEIL2 could rescue BER of uracil lesions driven by a dRP-deficient Pol β. The proficiency of NEIL1 in the full BER was higher compared with NEIL2, in agreement with the kinetic parameters showing that NEIL2 is the worst of the three dRPases. We have also reconstituted the repair of AP sites pre-formed in DNA by action of *E. coli* UDG. No major

Having established that NEIL1 and NEIL2 could substitute for dRPlyase activity of Pol β in the reconstituted BER system, we then studied whether NEIL proteins could manifest their

To identify proteins that have dRP lyase activity or influence removal of the dRP from BER intermediates in the absence of Pol β, we used Pol β null mouse embryonic fibroblast (MEF) cell extract for sodium borohydride driven cross-linking of the Schiff base dRP lyase intermediate protein-DNA complex (Prasad et al., 2007). The strong labeling of a single species in the Pol β null cell extracts corresponding to an unknown protein-DNA complex of

The preferential cross-linking of this protein reflected extraordinary specificity in light of the multitude of proteins in the cell. Taking into account the molecular masses of NEIL1 and NEIL2 (43.5 kDa and 38.2 kDa, respectively) the product could not be related to glycosylases. It should be noted that an apparent molecular mass of a covalent adduct protein-nucleic acid estimated by electrophoretic mobility is approximately equal to the sum

For identification of protein we applied the approach schematically depicted in Fig. 2. The DNA probe contained a 32P-labeled dRP moiety in a single-stranded break and a 3′-biotin tag to facilitate isolation of cross-linked protein-DNA complexes. Eleven of ions observed in MALDI MS spectrum corresponded to peptides of HMGB1. The (M + H)+ ion of m/z 1520.84 was selected automatically during the data dependent acquisition for MS/MS analysis. The values from both the peptide masses and the MS/MS fragment ion masses were used in a database search. The protein was identified as HMGB1 with a Mowse-based score of 102, 32% sequence coverage and a protein score confidence interval of 99.995%. Among the observed ions the ion of m/z 1520.84 corresponds to amino acid residues 113– 127 of the mouse HMGB1 and is a 'signature' that distinguishes HMGB1 from the closely related protein, HMGB2 (Bustin & Reeves, 1996). HMGB1 and HMGB2 are nuclear nonhistone DNA-binding proteins that belong to the high-mobility group box family of proteins (Bustin & Reeves, 1996). HMGB1 has an architectural role in the assembly of nucleoprotein complexes and is highly conserved across species (Bustin & Reeves, 1996; Tang et al., 2010 ;Liu et al., 2010; Stros, 2010). HMGB1 binds to DNA in the minor groove without sequence specificity and has the ability to transiently introduce bends or kinks into linear DNA (Liu et al., 2010; Stros, 2010). The intrinsic ability of HMGB1 to alter DNA structures allows it to participate in many biological processes including regulation of

dRPase activity in some particular systems, e.g. in cell extracts lacking Pol β.

**2.3.5 Identification of HMGB1 as cofactor of the BER process** 

of molecular masses of protein and attached nucleic acid.

difference from the repair of U was observed.

37 kDa was observed (Fig. 6A).

chromatin structure, transcription, DNA damage repair and recombination. The importance of HMGB1 in DNA repair was identified in studies that revealed the ability of HMGB1 to bind to a variety of bulky DNA lesions (Liu et al., 2010; Stros, 2010).

Fig. 6. Identification of HMGB1 as a BER cofactor (from Prasad et al., 2007). (A) Search of extract proteins interacting with the 5'dRP residue in the DNA duplex: lane *2*, products of cross-linking between 5' dRP DNA and MEF extract proteins expressing Pol β with flagepitope (FE), lane 1 control without borohydride treatment. (B) The influence of HMGB1 on FEN1 activity. (C) Influence of HMGB1 on APE1 activity. (D) Comparison of the 5' deoxyribose phosphate lyase activity of HMGB1 and Pol β. (E) Interaction of GFP–HMGB1 in HeLa cells with DNA damage sites induced by scanning laser microirradiation (λ 405 nm) without a sensitizer and in the presence of 8-methoxypsoralen (100 μM). Protein designation: 8-Oxoguanine DNA glycosylase (OGG1); NTH1, DNA glycosylase removing oxidized pyrimidines from DNA; RAD52, protein involved in double-strand break repair, homologous recombination; Ku70, Ku antigen subunit involved in of double-strand break repair, nonhomologous end joining. Arrows show the direction of the scan.

The observed ability of HMGB1 to interact with the BER DNA intermediate poses a question about its role in the process. It was found in the in vitro experiments that HMGB1 isolated from HeLa cells directly interacted with several BER proteins: APE1, Pol β, and FEN1( data not shown) and stimulate the activity of BER enzymes FEN1 and APE1 (Figs. 6B and 6C, respectively). HMGB1 was also revealed to have weak 5' dRP lyase activity (Fig. 6D). Using HeLa cells expressing HMGB1 in the form of a chimeric protein with green fluorescent protein (GFP–HMGB1), it was found that HMGB1 can be localized in the regions of DNA damage induced by laser microirradiation (Fig. 6E). Irradiation under used conditions generates both single-strand breaks and oxidized bases with high frequency (Lan et al., 2004). Indeed, DNA glycosylases (GFP–OGG1 and GFP–NTH1) efficiently accumulate

New Players in Recognition of Intact and Cleaved AP Sites:

substantially more resistant than parental cells.

Implication in DNA Repair in Mammalian Cells 319

generated. Then using transgenic and parental cell lines and employing a variety of cellbased assays and biochemical approaches, the authors provided evidence that the AP site/dRP lyase activities indeed had important biological functions. First, it has been demonstrated that HMGA2 could be efficiently trapped on genomic DNA. Parental cells A549, which express HMGA2 below detectable level, were exposed to low pH or physiological pH as control. DNA isolated from treated cells was incubated with recombinant HMGA2 under trapping (+NaCNBH3) or non-trapping (+NaCl) conditions. The subsequent dot-blot analysis revealed that HMGA2 could be only trapped by DNA derived from cells exposed to low pH, conditions leading to generation of AP sites. Moreover, HMGA2 expressed in transgenic cell line A549 (1.6) was efficiently trapped in a covalent complex *in vivo* with genomic AP sites generated when the cells were subjected to low pH. Analysis of cytotoxic effects that might result from depurination in parental and transgenic cells caused by exposure to low pH revealed that all transgenic cell lines were

In order to unravel the role of HMGA2 in response of cells to genotoxic impact, parental and transgenic cells were exposed to hydroxyurea (Hu) or methyl methanesulphonate (MMS). Hu is able to induce base oxidation and depurination (Sakano et al., 2001). MMS produces genomic AP sites through the action of DNA glycosylases, which remove the alkylated bases (Sedgwik et al., 2006). In the case of both reagents expression of HMGA2 resulted in

Both AP and dRP lyase activities play central roles in the early steps of BER (Hegde et al., 2008). In order to demonstrate that protection from MMS induced DNA damage observed with transgenic cells involves HMGA2 lyase activities the cells were sequentially exposed to MMS and O-benzyl hydroxylamine (BA). BA alone had no effect on the survival of parental or transgenic cells. However, combined action of MMS and BA sensitized HMGA2-containg cells to MMS treatment. BA (analogously to methoxy amine) reacts with the baseless deoxyribose moieties (in intact or cleaved AP sites) rendering them refractory to

Direct interaction of HMGA2 with APE1 *in vitro* and *in vivo* (Sgarra et al., 2008, Summer et al., 2009, Sgarra et al., 2010) has been reported. However, the influence of HMGA2 on the AP endonuclease 1 activity is still unknown. HMGA2 protects cells against three different genotoxicants, i.e. Hu, MMS and low pH (Summer et al., 2009), which introduce the DNA damages repaired by the BER machinery. It is noteworthy here, that repair of these lesions involves the common intermediates, AP sites and 5' dRP moieties, which can be processed by HMGA2. This strongly support the idea developed in (Summer et al., 2009) that intrinsic AP/dRP lyase activities of HMGA2 are responsible for the protective action of this protein. However, one could not exclude that in addition to direct action, HMGA2 influences the BER capacity indirectly by enhancing the activity of APE1 as was observed in the case of HMGB1 (Prasad et al., 2007). Activation of APE1 by protein-protein interaction may be involved both in the stage of AP site hydrolysis and removing the 3' end PUA group. APE1 is known as the main mammalian protein capable to excise this blocking group producing

Methylated bases in DNA generated by endogenous and environmental alkylating agents can be removed by three distinct strategies. While 3-methyladenine (3-alkyladenine) is

significant protection against cell death leading to increase in cell survival.

mammalian AP endonuclease 1 and AP/dRP lyase activities (Horton et al., 2000).

the 3' end hydroxyl moiety (Wilson & Barsky, 2001; Pascucci et al., 2002).

**2.3.7 Human ALK B homologue (ABH1) is an AP lyase** 

in sites of irradiation unlike the proteins recognizing double-strand breaks in DNA (GFP– Ku70 and GFP–RAD52) (Fig. 6E).

Mouse embryonic fibroblasts of the HMGB1+/+ type are more sensitive than HMGB1–/– cells to the combined action of methyl methanesulfonate and methoxyamine, and HMGB1+/+ cells contain a much larger amount of single-strand breaks. The treatment of AP DNA with methoxyamine increases its resistance to APE1 action (Horton et al., 2000).

Another group of researches using two cultivated cell lines of breast cancer found that the increase in the expression level of HMGB1 alters the cells' phenotype by slowing cell growth and increasing the cell sensitivity to ionizing radiation (Jiao et al., 2007).

Interestingly, that in spite of ability of purified NEIL 1/2 to interact with dRP lyase substrate (Grin et al., 2006) we did not reveal abundant products of their cross-linking in the Pol β null MEF extract (Fig. 6 A, lane 2). This interaction appears to be counteracted by effective binding of HMGB1, which is highly abundant in cells.

#### **2.3.6 HMGA as cofactor of the BER process**

It is interesting to note that dRP- and AP lyase activities were revealed for another group of chromatin proteins (Summer at al., 2009). Mammalian high mobility group proteins are nonhistone chromatin architectural factors encoded by two genes, *HMGA1* and *HMGA2*. Alternative mRNA splicing results in at least four protein isoforms involved in chromatin remodeling and gene transcription (Bustin & Reeves, 1996, Reeves, 2001, Cleynen & van de Ven, 2008). HMGA proteins are characterized by the presence of an acidic C-terminal tail and three DNA binding domains containing short basic repeats, the so called AT-hooks, capable to bind in the minor groove of AT-rich sequences in DNA. In humans, HMGA expression is undetectable or very low in differentiated adult tissues, but high HMGA protein levels are associated with human malignant neoplasias (Berner et al., 1997, Abe et al., 2003, Miyazawa et al., 2004, Meyer et al., 2007). In addition, expression of HMGA1 is functionally linked to chemoresistance of some human carcinomas (Liau & Whang, 2008).

Recombinant human HMGA (HMGA1a, HMGA1b and HMGA2) proteins have been shown to efficiently cleave plasmid DNA containing AP sites (Summer et al., 2009). Further analysis revealed that the proteins could be trapped on AP DNA by NaCNBH3 treatment, the mechanism characteristic of AP lyase activity (David & Williams, 1998; McCullough et al.; 1999, Piersen et al., 2000). To determine the chemical nature of DNA ends generated by the HMGA proteins and the efficiency of AP site cleavage, 32P-labeled double-stranded short DNA duplex containing a single AP site was used as substrate. The analysis revealed that HMGA proteins generated cleavage products, which exhibit the same electrophoretic mobility as those produced by endonuclease III of *E. coli*, an AP lyase catalyzing the β elimination reaction (McCullough et al., 1999; David & Williams, 1998).

To test the possibility that HMGA proteins also possess the related 5′-dRP lyase activity the same DNA duplex bearing label at the 3′ end was employed. A 5′-dRP moiety on the labeled strand was produced by endonuclease IV from *E. coli.* To stabilize chemically labile 5′-dRP group and to improve electrophoretic separation of the products, 5′-deoxyribosyl phosphate moiety was adducted with O-benzyl hydroxylamine. The analysis revealed that the HMGA proteins efficiently removed 5′-dRP moiety. Thus, HMGA proteins display the AP/5′-dRP lyase activity characteristic of the BER process.

Having established that the HMGA proteins are lyases, the authors examined the role of this activity in cell context. To this end, cell lines constitutively expressing HMGA2 have been

in sites of irradiation unlike the proteins recognizing double-strand breaks in DNA (GFP–

Mouse embryonic fibroblasts of the HMGB1+/+ type are more sensitive than HMGB1–/– cells to the combined action of methyl methanesulfonate and methoxyamine, and HMGB1+/+ cells contain a much larger amount of single-strand breaks. The treatment of AP DNA with methoxyamine increases its resistance to APE1 action (Horton et al., 2000). Another group of researches using two cultivated cell lines of breast cancer found that the increase in the expression level of HMGB1 alters the cells' phenotype by slowing cell growth

Interestingly, that in spite of ability of purified NEIL 1/2 to interact with dRP lyase substrate (Grin et al., 2006) we did not reveal abundant products of their cross-linking in the Pol β null MEF extract (Fig. 6 A, lane 2). This interaction appears to be counteracted by effective

It is interesting to note that dRP- and AP lyase activities were revealed for another group of chromatin proteins (Summer at al., 2009). Mammalian high mobility group proteins are nonhistone chromatin architectural factors encoded by two genes, *HMGA1* and *HMGA2*. Alternative mRNA splicing results in at least four protein isoforms involved in chromatin remodeling and gene transcription (Bustin & Reeves, 1996, Reeves, 2001, Cleynen & van de Ven, 2008). HMGA proteins are characterized by the presence of an acidic C-terminal tail and three DNA binding domains containing short basic repeats, the so called AT-hooks, capable to bind in the minor groove of AT-rich sequences in DNA. In humans, HMGA expression is undetectable or very low in differentiated adult tissues, but high HMGA protein levels are associated with human malignant neoplasias (Berner et al., 1997, Abe et al., 2003, Miyazawa et al., 2004, Meyer et al., 2007). In addition, expression of HMGA1 is functionally linked to chemoresistance of some human carcinomas (Liau & Whang, 2008). Recombinant human HMGA (HMGA1a, HMGA1b and HMGA2) proteins have been shown to efficiently cleave plasmid DNA containing AP sites (Summer et al., 2009). Further analysis revealed that the proteins could be trapped on AP DNA by NaCNBH3 treatment, the mechanism characteristic of AP lyase activity (David & Williams, 1998; McCullough et al.; 1999, Piersen et al., 2000). To determine the chemical nature of DNA ends generated by the HMGA proteins and the efficiency of AP site cleavage, 32P-labeled double-stranded short DNA duplex containing a single AP site was used as substrate. The analysis revealed that HMGA proteins generated cleavage products, which exhibit the same electrophoretic mobility as those produced by endonuclease III of *E. coli*, an AP lyase catalyzing the β

and increasing the cell sensitivity to ionizing radiation (Jiao et al., 2007).

elimination reaction (McCullough et al., 1999; David & Williams, 1998).

lyase activity characteristic of the BER process.

To test the possibility that HMGA proteins also possess the related 5′-dRP lyase activity the same DNA duplex bearing label at the 3′ end was employed. A 5′-dRP moiety on the labeled strand was produced by endonuclease IV from *E. coli.* To stabilize chemically labile 5′-dRP group and to improve electrophoretic separation of the products, 5′-deoxyribosyl phosphate moiety was adducted with O-benzyl hydroxylamine. The analysis revealed that the HMGA proteins efficiently removed 5′-dRP moiety. Thus, HMGA proteins display the AP/5′-dRP

Having established that the HMGA proteins are lyases, the authors examined the role of this activity in cell context. To this end, cell lines constitutively expressing HMGA2 have been

binding of HMGB1, which is highly abundant in cells.

**2.3.6 HMGA as cofactor of the BER process** 

Ku70 and GFP–RAD52) (Fig. 6E).

generated. Then using transgenic and parental cell lines and employing a variety of cellbased assays and biochemical approaches, the authors provided evidence that the AP site/dRP lyase activities indeed had important biological functions. First, it has been demonstrated that HMGA2 could be efficiently trapped on genomic DNA. Parental cells A549, which express HMGA2 below detectable level, were exposed to low pH or physiological pH as control. DNA isolated from treated cells was incubated with recombinant HMGA2 under trapping (+NaCNBH3) or non-trapping (+NaCl) conditions. The subsequent dot-blot analysis revealed that HMGA2 could be only trapped by DNA derived from cells exposed to low pH, conditions leading to generation of AP sites. Moreover, HMGA2 expressed in transgenic cell line A549 (1.6) was efficiently trapped in a covalent complex *in vivo* with genomic AP sites generated when the cells were subjected to low pH. Analysis of cytotoxic effects that might result from depurination in parental and transgenic cells caused by exposure to low pH revealed that all transgenic cell lines were substantially more resistant than parental cells.

In order to unravel the role of HMGA2 in response of cells to genotoxic impact, parental and transgenic cells were exposed to hydroxyurea (Hu) or methyl methanesulphonate (MMS). Hu is able to induce base oxidation and depurination (Sakano et al., 2001). MMS produces genomic AP sites through the action of DNA glycosylases, which remove the alkylated bases (Sedgwik et al., 2006). In the case of both reagents expression of HMGA2 resulted in significant protection against cell death leading to increase in cell survival.

Both AP and dRP lyase activities play central roles in the early steps of BER (Hegde et al., 2008). In order to demonstrate that protection from MMS induced DNA damage observed with transgenic cells involves HMGA2 lyase activities the cells were sequentially exposed to MMS and O-benzyl hydroxylamine (BA). BA alone had no effect on the survival of parental or transgenic cells. However, combined action of MMS and BA sensitized HMGA2-containg cells to MMS treatment. BA (analogously to methoxy amine) reacts with the baseless deoxyribose moieties (in intact or cleaved AP sites) rendering them refractory to mammalian AP endonuclease 1 and AP/dRP lyase activities (Horton et al., 2000).

Direct interaction of HMGA2 with APE1 *in vitro* and *in vivo* (Sgarra et al., 2008, Summer et al., 2009, Sgarra et al., 2010) has been reported. However, the influence of HMGA2 on the AP endonuclease 1 activity is still unknown. HMGA2 protects cells against three different genotoxicants, i.e. Hu, MMS and low pH (Summer et al., 2009), which introduce the DNA damages repaired by the BER machinery. It is noteworthy here, that repair of these lesions involves the common intermediates, AP sites and 5' dRP moieties, which can be processed by HMGA2. This strongly support the idea developed in (Summer et al., 2009) that intrinsic AP/dRP lyase activities of HMGA2 are responsible for the protective action of this protein. However, one could not exclude that in addition to direct action, HMGA2 influences the BER capacity indirectly by enhancing the activity of APE1 as was observed in the case of HMGB1 (Prasad et al., 2007). Activation of APE1 by protein-protein interaction may be involved both in the stage of AP site hydrolysis and removing the 3' end PUA group. APE1 is known as the main mammalian protein capable to excise this blocking group producing the 3' end hydroxyl moiety (Wilson & Barsky, 2001; Pascucci et al., 2002).

### **2.3.7 Human ALK B homologue (ABH1) is an AP lyase**

Methylated bases in DNA generated by endogenous and environmental alkylating agents can be removed by three distinct strategies. While 3-methyladenine (3-alkyladenine) is

New Players in Recognition of Intact and Cleaved AP Sites:

Implication in DNA Repair in Mammalian Cells 321

Fig. 7. Comparison of product sizes for ABH1, APE1, ENDOIII, and ENDOVIII, and examination of the effect of phosphatase treatment (from Müller et al., 2010). The uracil containing DNA duplex was cleaved by incubation with ABH1 or the control endonucleases in the presence of UDG for 1 h at 37 ºC. Portions of the samples were treated with the phosphatase T4 polynucleotide kinase to remove possible phosphates at the 3' end of the labeled products. The presence of a 3'-phosphate causes the oligonucleotide to migrate more rapidly than the non-phosphorylated species due to the extra negative charge; removal of the phosphate results in a shift to an apparently larger product, as seen with EndoVIII.

Taking into account ubiquitous expression of APE1 and inefficiency of the ABH1 AP lyase activity, ABH1 hardly play significant role *in vivo* in processing of AP sites. Moreover, the ability of ABH1 to more efficiently cleave opposite AP sites in ds DNA that may result in formation of DS breaks, more toxic for the cells than AP sites, therefore action of ABH1 on clustered AP sites in genomic DNA appears to be dangerous for cells. The authors considered tight binding of ABH1 with the product as a mechanism that protects ends from degradation. On the other hand, tight binding may create hindrances for the repair processes and require special efforts to remove blocking group from the 3' end. Potentially interesting finding of intrinsic AP lyase activity of ABH1 requires additional studies to draw

**2.3.8 Tyrosyl-DNA-phosphodiesterase mediates the new APE-independent BER** 

As mentioned above AP sites can be cleaved by activity of bifunctional DNA glycosylases with associated AP lyase activities via β- or β,δ-elimination mechanism producing DNA intermediates with 3' end containing 3'-phosphate or 3'-PUA groups (Fig. 1) that have to be removed prior to DNA synthesis may occur. DNA intermediates with blocked 3'end may also appear from action of ROS and as a result of spontaneous decomposition of AP sites. The 3' PUA is known to be removed by the only AP-endonuclease, APE1, which possesses 3' phosphodiesterase activity with α-unsaturated aldehydes, producing a single nucleotide gap flanked by a 3'-hydroxyl group and a 5' phosphate group (Wilson & Barsky, 2001;

a conclusion concerning significance of discovered activity *in vivo.* 

**pathway in mammals** 

excised by a specific DNA glycosylase that initiates a base excision repair process, 1 methyladenine, 3-methylcytosine and O6-methylguanine are corrected by direct reversal exploring a different mechanism (for more information see a review Sedgwick et al., 2006). One of the strategies of direct reversal involves the activity of DNA dioxygenases, which release the methyl moiety as formaldehyde (Duncan et al., 2002). Although three human DNA dioxygenases – ABH1–ABH3 – catalyze the same oxidative demethylation reaction they display specificity toward methylated base and nucleic acids (DNA or RNA and singleor double-stranded) (Duncan et al., 2002, Westbye et al., 2008, Ougland et al., 2004; Kurowski et al., 2003). Unexpectedly, ABH1 – the closest AlkB *E. coli* homologue – has been shown to display an AP site cleavage activity (Müller et al., 2010).

Intensive study of discovered activity revealed that the DNA cleavage activity of ABH1 did not require added Fe2+ or 2-oxoglutarate, is not inhibited by EDTA, and is unaffected by mutation of the putative metal-binding residues, indicating that this activity arises from an active site distinct from that used for demethylation.

Enzymes that cleave sugar-phosphate backbone at abasic sites can utilize hydrolysis, β- or β,δ-elimination mechanisms (Fig. 1). First, to assess the cleavage mechanism, the activity of ABH1 was examined with DNA containing THF residue, the AP-site analogue, which could not be cleaved by the β-elimination reaction. No AP site cleavage was observed with ABH1 and EndoIII unlike APE1. Second, the electrophoretic mobility of the products resulting from the activities of ABH1, APE1, EndoIII and EndoVIII were examined. Prior to analysis 5′-[32P]-labeled ds-oligonucleotides containing the AP site were incubated with the corresponding enzymes. In some samples, the products of AP site cleavage were additionally treated with T4 polynucleotide kinase (PNK) to remove possible 3′-terminal phosphate by this phosphatase. It should be noted that authors used proteinase treatment of the reaction mixtures to stop the reaction and degrade the enzymes prior to separation of oligonucleotides by denaturing PAAG electrophoresis (Fig. 7). The products produced by ABH1 migrate slowly (Fig. 7, lane 1) than the product of the β-elimination reaction (Fig. 7, lane 3) and do not contain the 3′ end phosphate group since the mobility of the products was not changed by PNK treatment (Fig. 7, compare lanes 1 and 2). While the products derived from the EndoVIII activity, which explores the β,δ-elimination mechanism resulting in the 3' phosphate group, migrate slowly after PNK treatment (Fig. 7, compare lanes 5 and 6). The authors proposed that ABH1 cleaves AP sites by β-elimination with ABH1 being bound with the product in tight complex. They attribute the slight decrease in mobility of the products for the ABH1 samples to tight binding of ABH1 fragments with oligonucleotides. The authors demonstrated that both ABH1 and EndoIII in the presence of NaBH4 are able to generate stable products with single-stranded AP-DNA, double-stranded DNA containing one AP site and double-stranded DNA containing two AP sites, but the important control without reducing agent is missed. Taking into account that ABH1 forms stable adducts with AP DNA without reduction, as observed in the activity test (Fig. 7, lanes 1 and 2), it is questionable whether the trapping of ABH1 is the Schiff base dependent.

ABH1 was shown to display specificity in substrate usage with DNA containing two AP sites being the preferable substrate. Further analysis of AP site cleavage activity at different substrate-to-enzyme ratio demonstrated that concentration of product was always substoichiometric to the enzyme concentration that is in agreement with tight binding of ABH1 with the product.

excised by a specific DNA glycosylase that initiates a base excision repair process, 1 methyladenine, 3-methylcytosine and O6-methylguanine are corrected by direct reversal exploring a different mechanism (for more information see a review Sedgwick et al., 2006). One of the strategies of direct reversal involves the activity of DNA dioxygenases, which release the methyl moiety as formaldehyde (Duncan et al., 2002). Although three human DNA dioxygenases – ABH1–ABH3 – catalyze the same oxidative demethylation reaction they display specificity toward methylated base and nucleic acids (DNA or RNA and singleor double-stranded) (Duncan et al., 2002, Westbye et al., 2008, Ougland et al., 2004; Kurowski et al., 2003). Unexpectedly, ABH1 – the closest AlkB *E. coli* homologue – has been

Intensive study of discovered activity revealed that the DNA cleavage activity of ABH1 did not require added Fe2+ or 2-oxoglutarate, is not inhibited by EDTA, and is unaffected by mutation of the putative metal-binding residues, indicating that this activity arises from an

Enzymes that cleave sugar-phosphate backbone at abasic sites can utilize hydrolysis, β- or β,δ-elimination mechanisms (Fig. 1). First, to assess the cleavage mechanism, the activity of ABH1 was examined with DNA containing THF residue, the AP-site analogue, which could not be cleaved by the β-elimination reaction. No AP site cleavage was observed with ABH1 and EndoIII unlike APE1. Second, the electrophoretic mobility of the products resulting from the activities of ABH1, APE1, EndoIII and EndoVIII were examined. Prior to analysis 5′-[32P]-labeled ds-oligonucleotides containing the AP site were incubated with the corresponding enzymes. In some samples, the products of AP site cleavage were additionally treated with T4 polynucleotide kinase (PNK) to remove possible 3′-terminal phosphate by this phosphatase. It should be noted that authors used proteinase treatment of the reaction mixtures to stop the reaction and degrade the enzymes prior to separation of oligonucleotides by denaturing PAAG electrophoresis (Fig. 7). The products produced by ABH1 migrate slowly (Fig. 7, lane 1) than the product of the β-elimination reaction (Fig. 7, lane 3) and do not contain the 3′ end phosphate group since the mobility of the products was not changed by PNK treatment (Fig. 7, compare lanes 1 and 2). While the products derived from the EndoVIII activity, which explores the β,δ-elimination mechanism resulting in the 3' phosphate group, migrate slowly after PNK treatment (Fig. 7, compare lanes 5 and 6). The authors proposed that ABH1 cleaves AP sites by β-elimination with ABH1 being bound with the product in tight complex. They attribute the slight decrease in mobility of the products for the ABH1 samples to tight binding of ABH1 fragments with oligonucleotides. The authors demonstrated that both ABH1 and EndoIII in the presence of NaBH4 are able to generate stable products with single-stranded AP-DNA, double-stranded DNA containing one AP site and double-stranded DNA containing two AP sites, but the important control without reducing agent is missed. Taking into account that ABH1 forms stable adducts with AP DNA without reduction, as observed in the activity test (Fig. 7, lanes 1 and 2), it is

shown to display an AP site cleavage activity (Müller et al., 2010).

questionable whether the trapping of ABH1 is the Schiff base dependent.

with the product.

ABH1 was shown to display specificity in substrate usage with DNA containing two AP sites being the preferable substrate. Further analysis of AP site cleavage activity at different substrate-to-enzyme ratio demonstrated that concentration of product was always substoichiometric to the enzyme concentration that is in agreement with tight binding of ABH1

active site distinct from that used for demethylation.

Fig. 7. Comparison of product sizes for ABH1, APE1, ENDOIII, and ENDOVIII, and examination of the effect of phosphatase treatment (from Müller et al., 2010). The uracil containing DNA duplex was cleaved by incubation with ABH1 or the control endonucleases in the presence of UDG for 1 h at 37 ºC. Portions of the samples were treated with the phosphatase T4 polynucleotide kinase to remove possible phosphates at the 3' end of the labeled products. The presence of a 3'-phosphate causes the oligonucleotide to migrate more rapidly than the non-phosphorylated species due to the extra negative charge; removal of the phosphate results in a shift to an apparently larger product, as seen with EndoVIII.

Taking into account ubiquitous expression of APE1 and inefficiency of the ABH1 AP lyase activity, ABH1 hardly play significant role *in vivo* in processing of AP sites. Moreover, the ability of ABH1 to more efficiently cleave opposite AP sites in ds DNA that may result in formation of DS breaks, more toxic for the cells than AP sites, therefore action of ABH1 on clustered AP sites in genomic DNA appears to be dangerous for cells. The authors considered tight binding of ABH1 with the product as a mechanism that protects ends from degradation. On the other hand, tight binding may create hindrances for the repair processes and require special efforts to remove blocking group from the 3' end. Potentially interesting finding of intrinsic AP lyase activity of ABH1 requires additional studies to draw a conclusion concerning significance of discovered activity *in vivo.* 

#### **2.3.8 Tyrosyl-DNA-phosphodiesterase mediates the new APE-independent BER pathway in mammals**

As mentioned above AP sites can be cleaved by activity of bifunctional DNA glycosylases with associated AP lyase activities via β- or β,δ-elimination mechanism producing DNA intermediates with 3' end containing 3'-phosphate or 3'-PUA groups (Fig. 1) that have to be removed prior to DNA synthesis may occur. DNA intermediates with blocked 3'end may also appear from action of ROS and as a result of spontaneous decomposition of AP sites.

The 3' PUA is known to be removed by the only AP-endonuclease, APE1, which possesses 3' phosphodiesterase activity with α-unsaturated aldehydes, producing a single nucleotide gap flanked by a 3'-hydroxyl group and a 5' phosphate group (Wilson & Barsky, 2001;

New Players in Recognition of Intact and Cleaved AP Sites:

termini that expands the ability of the BER process.

(Caldecott, 2003).

site (Fig. 9B, lanes 4 and 6).

ligase III (Mani et al., 2004; Caldecott, 2003; Whitehouse et al., 2001).

**2.3.9 XRCC1 interactions with base excision repair DNA intermediates** 

Implication in DNA Repair in Mammalian Cells 323

minimal reconstituted BER system consisting of purified proteins (Fig. 8B). The 5′ 32Plabeled 32-mer DNA duplex containing uridine at the position 16 was incubated with the purified recombinant UDG, Tdp1, PNKP, Pol β, DNA ligase III, and XRCC1 to mimic DNA repair system. The reaction mixture containing Tdp1 but lacking PNKP (lane 2) generated a product with a 3′-phosphate, which is identical to that produced by NEIL1. Addition of PNKP resulted in a 15-mer product with the 3′-OH termini (lane 3). XRCC1, a scaffold protein, stimulates the activity of BER proteins, such as DNA polymerase β, PNKP, DNA

Lastly, DNA polymerase β replaces the missing DNA segment (lane 4) and DNA ligase reseals the DNA (lane 5). So, the repair of AP site initiated by Tdp1 fully restored the intact DNA and generated the products of the expected lengths at each intermediate stage. In summary, human Tdp1 protein can initiate APE1-independent repair of AP sites and 3′ PUA

XRCC1 is known to play a crucial role in the coordination of two overlapping repair pathways, SSBR and BER (Caldecott, 2003). Although the main role of XRCC1 during BER has been attributed to its participation in the post-incision steps (Wong et al., 2005), which are shared with SSBR, the physical and functional interactions with proteins involved in the initiation of modified bases or abasic sites repair (Marsin et al., 2003, Campalans et al., 2005, Vidal et al., 2001, Wiederhold et al., 2004) suggest that XRCC1 presence at the early steps of BER could be important for assuring a correct repair process. One possible role of XRCC1 could be to optimize the passage of DNA substrates from one enzyme to the next one in the pathway by holding the proteins together through its different interacting domains

Investigating the interactions of with different BER DNA intermediates generated either by DNA glycosylase hOGG1 or AP endonuclease APE1 we have found that XRCC1 is able to interact with AP sites via formation of the Schiff base intermediate (Nazarkina et al., 2007). Because hOGG1 possesses both, DNA glycosylase and AP lyase activities, either of two DNA intermediates can be produced: DNA duplex containing an intact AP site or a nick with a 3′ PUA moiety (See Fig. 1). By competition experiments using the THF-containing or regular DNA duplex it was demonstrated that XRCC1 binds DNA with an AP site, or its

XRCC1 is known to bind DNA with single-strand breaks with higher affinity that regular DNA duplex (Mani et al., 2004). We then investigated the relative affinities of XRCC1 to different DNA structures that could be BER intermediates in the repair of single or double DNA lesions. The efficiency of cross-linking is maximal with an incised AP site 3' PUA (Fig. 9A, lane 3). Interestingly, the presence of a strand interruption on the complementary oligonucleotide strongly stimulated the cross-linking of XRCC1 to the AP site. These results suggest that XRCC1 could be important to hold the DNA together during the repair of clustered DNA damage. XRCC1 is also able to cross-link to a 5' dRP residue downstream of a nick (Fig. 9A, lane 6). Comparative analysis of the patterns of protein cross-linking to AP DNA in cell extracts deficient (EM9) and proficient in XRCC1 (EM9-X) (Fig. 9B, lanes 3–6) revealed the product that can be related with XRCC1 (lane 6). Interestingly, that the band is observed with DNA containing interruption opposite AP

synthetic analogue, with considerably higher affinity than regular DNA duplex.

Pascucci et al., 2002). However, APE1 is barely active in removing 3' phosphate generated by mammalian DNA glycosylases NEIL1 and NEIL2. The 3' phosphate is efficiently removed by polynucleotide kinase (PNK) and not APE1 (Wiederhold et al., 2004).

Tyrosyl-DNA phosphodiesterase (Tdp1) was discovered as an enzymatic activity from *Saccharomyces cerevisiae* that specifically hydrolyzes the phosphodiester linkage between the O-4 atom of a tyrosine and a DNA 3′ phosphate (Yang et al., 1996). This type of linkage is typical for the covalent reaction intermediate produced upon Topoisomerase 1 cleavage of one DNA strand. Human Tdp1 can also hydrolyze other 3′-end DNA alterations that are covalently linked to the DNA, indicating that it may function as a general 3′-DNA phosphodiesterase and repair enzyme (Dexheimer et al., 2008). Tdp1 can also remove the tetrahydrofuran moiety from the 3'-end of DNA (Interthal et al., 2005). It is conceivable that Tdp1 acts on the 3′ PUA moiety.

To study an ability of Tdp1 to process the 3′ PUA moiety AP DNA was first incubated with Endo III (Fig. 8A, lane 2) (Lebedeva et al., 2011). Following incubation of this product with Tdp1 results in 15-mer with 3' phosphate, which can be removed by PNKP (Fig. 8A, lane 3). Thus, Tdp1 is able to remove the 3′ PUA that allows to realize the APE1-independent pathway of BER where AP sites are cleaved by bifunctional DNA glycosylases via the βelimination mechanism.

Fig. 8. Tdp1 activity on DNA substrate with 3'-dRP moiety in the single strand break (A) and reconstitution of the AP-DNA substrate repair initiated by Tdp1 (B) (From Lebedeva et al., 2011)**. (A)** The 15-mer with 3'-dRP was generated by incubation of AP DNA with EndoIII (lane 2). Following incubation of this product with Tdp1 results in 15-mer with 3'-P (lane 3). Lane 4 shows the 15-mer product with 3'-OH after adding PNKP in the reaction mixture. Lane 1 corresponds to the AP-DNA substrate incubated with UDG. Lane 5 - control. (B) 5' end labeled AP-DNA substrate was subsequently incubated with the UDG (lane 1), Tdp1 (lane 2), PNKP and XRCC1 (lane 3), Pol β (lane 4), DNA ligase III (lane 5). The components present in different reaction mixtures are indicated.

We tested an ability of Tdp1 to process AP sites (natural or mimicked by THF residue) and found that Tdp1 cleaves both types of AP sites generating the product identical to the product of β,δ-elimination (data not shown). Finally, the repair of AP site was analyzed in a

Pascucci et al., 2002). However, APE1 is barely active in removing 3' phosphate generated by mammalian DNA glycosylases NEIL1 and NEIL2. The 3' phosphate is efficiently removed

Tyrosyl-DNA phosphodiesterase (Tdp1) was discovered as an enzymatic activity from *Saccharomyces cerevisiae* that specifically hydrolyzes the phosphodiester linkage between the O-4 atom of a tyrosine and a DNA 3′ phosphate (Yang et al., 1996). This type of linkage is typical for the covalent reaction intermediate produced upon Topoisomerase 1 cleavage of one DNA strand. Human Tdp1 can also hydrolyze other 3′-end DNA alterations that are covalently linked to the DNA, indicating that it may function as a general 3′-DNA phosphodiesterase and repair enzyme (Dexheimer et al., 2008). Tdp1 can also remove the tetrahydrofuran moiety from the 3'-end of DNA (Interthal et al., 2005). It is conceivable that

To study an ability of Tdp1 to process the 3′ PUA moiety AP DNA was first incubated with Endo III (Fig. 8A, lane 2) (Lebedeva et al., 2011). Following incubation of this product with Tdp1 results in 15-mer with 3' phosphate, which can be removed by PNKP (Fig. 8A, lane 3). Thus, Tdp1 is able to remove the 3′ PUA that allows to realize the APE1-independent pathway of BER where AP sites are cleaved by bifunctional DNA glycosylases via the β-

Fig. 8. Tdp1 activity on DNA substrate with 3'-dRP moiety in the single strand break (A) and reconstitution of the AP-DNA substrate repair initiated by Tdp1 (B) (From Lebedeva et al., 2011)**. (A)** The 15-mer with 3'-dRP was generated by incubation of AP DNA with EndoIII (lane 2). Following incubation of this product with Tdp1 results in 15-mer with 3'-P (lane 3). Lane 4 shows the 15-mer product with 3'-OH after adding PNKP in the reaction mixture. Lane 1 corresponds to the AP-DNA substrate incubated with UDG. Lane 5 - control. (B) 5' end labeled AP-DNA substrate was subsequently incubated with the UDG (lane 1), Tdp1 (lane 2), PNKP and XRCC1 (lane 3), Pol β (lane 4), DNA ligase III (lane 5). The components

We tested an ability of Tdp1 to process AP sites (natural or mimicked by THF residue) and found that Tdp1 cleaves both types of AP sites generating the product identical to the product of β,δ-elimination (data not shown). Finally, the repair of AP site was analyzed in a

present in different reaction mixtures are indicated.

by polynucleotide kinase (PNK) and not APE1 (Wiederhold et al., 2004).

Tdp1 acts on the 3′ PUA moiety.

elimination mechanism.

minimal reconstituted BER system consisting of purified proteins (Fig. 8B). The 5′ 32Plabeled 32-mer DNA duplex containing uridine at the position 16 was incubated with the purified recombinant UDG, Tdp1, PNKP, Pol β, DNA ligase III, and XRCC1 to mimic DNA repair system. The reaction mixture containing Tdp1 but lacking PNKP (lane 2) generated a product with a 3′-phosphate, which is identical to that produced by NEIL1. Addition of PNKP resulted in a 15-mer product with the 3′-OH termini (lane 3). XRCC1, a scaffold protein, stimulates the activity of BER proteins, such as DNA polymerase β, PNKP, DNA ligase III (Mani et al., 2004; Caldecott, 2003; Whitehouse et al., 2001).

Lastly, DNA polymerase β replaces the missing DNA segment (lane 4) and DNA ligase reseals the DNA (lane 5). So, the repair of AP site initiated by Tdp1 fully restored the intact DNA and generated the products of the expected lengths at each intermediate stage. In summary, human Tdp1 protein can initiate APE1-independent repair of AP sites and 3′ PUA termini that expands the ability of the BER process.
