**2.3.4 New function of Neil1 and Neil2 as 5′dRP lyase**

According to current view, in mammalian BER the sub-pathway choice is influenced by the rate-limiting step in SN BER, i.e., removal of the 5'-dRP by the dRP lyase activity of Pol β (Horton et al., 2000; Srivastava et al., 1998). For example, if the 5'-dRP cannot be removed efficiently, continued DNA synthesis will emphasize the LP BER sub-pathway (Horton et al., 2000). Yet, both subpathways appear to operate simultaneously to repair most types of DNA lesions *in vitro* (Horton et al., 2000; Prasad et al., 2000). It has been shown previously that the 5'-dRP BER intermediate is the cytotoxic lesion following exposure to methylating agents, and its removal is a pivotal step in BER *in vivo* (Sobol et al., 2000).

Pol β-deficient mouse cells show little dRPase activity (Sobol et al., 2000), but some residual dRP removal by extracts prepared from these cells is still present (Podlutsky et al., 2001). It is possible that while Pol β carries out the bulk of dRP removal from DNA, other activities could be more specifically employed for certain lesions, cell or tissue types, or at certain points of the cell cycle. In *E. coli*, Fpg (formamidopyrimidine-DNA glycosylase) and to a lesser extent endonuclease VIII (Nei) catalyze β-elimination of dRP moiety (Fig. 1). Three mammalian homologues of bacterial Fpg and Nei termed NEIL (Nei-like)-1, -2, and -3 have been identified (Hazra et al., 2002; Hegde et al., 2008). Based on the similarity of their active sites to those of Fpg and Nei, one could expect that they could also display dRPase activity. We have shown that two of these proteins, NEIL1 and NEIL2, are capable of removing dRP lesions from DNA with the efficiency comparable to that of Pol β, and that they can substitute for Pol β dRPase activity in a reconstituted BER assay (Grin et al., 2006).

dRPase activity can be revealed with 3'-labeled nicked abasic oligonucleotide substrates. Such substrates were prepared by end-filling of a 5'-overhanging oligonucleotide duplexes with 32P-labeled dATP and the consecutive treatment of the duplex with uracil DNA glycosylase (Ung) and APE1. The resulting dRP site is unstable in nucleophilic buffers and is degraded during migration through Tris-containing polyacrylamide gels, therefore the products were stabilized by sodium borohydride reduction immediately after the dRPasecatalyzed reaction.

Fig. 5A illustrates that both NEIL1 and NEIL2 possess a dRP-removing activity. The dRPase activities of NEIL1 and NEIL2 demonstrated the enzyme concentration and time dependence expected of an enzyme-catalyzed reaction (Fig. 5B and data not shown). Notably, the activity of NEIL1 in these experiments appeared higher than that of NEIL1 (Fig. 5B). Both NEIL1 and NEIL2 excised with similar efficiency when A, C, or T were placed opposite the lesion, and the excision of dRP opposite G was 1.5–2-fold lower; Pol β removed dRP equally well from all opposite-base contexts. To confirm that dRP removal by NEIL1 and NEIL2 proceeds by β-elimination, as in Pol β and Fpg, we have performed the reaction in the presence of sodium borohydride, which reduces the Schiff base covalent complexes formed between the catalytic amine nucleophile of dRP lyases and C1' of the dRP site during the reaction (Fig. 5C). Such trapped enzyme–DNA complexes are stable enough to be

Thus, by virtue of PARP-1's ability to interact with the intact AP sites (single or within cluster) via Schiff base formation, we demonstrated a new role for PARP-1 in regulation of the BER process. PARP-1's interaction at the AP site could recruit this key enzyme and protect the site until APE1 becomes available to initiate strand incision and BER. Alternatively, PARP-1's 5'-dRP/AP lyase activity could perform strand incision and trigger

According to current view, in mammalian BER the sub-pathway choice is influenced by the rate-limiting step in SN BER, i.e., removal of the 5'-dRP by the dRP lyase activity of Pol β (Horton et al., 2000; Srivastava et al., 1998). For example, if the 5'-dRP cannot be removed efficiently, continued DNA synthesis will emphasize the LP BER sub-pathway (Horton et al., 2000). Yet, both subpathways appear to operate simultaneously to repair most types of DNA lesions *in vitro* (Horton et al., 2000; Prasad et al., 2000). It has been shown previously that the 5'-dRP BER intermediate is the cytotoxic lesion following exposure to methylating

Pol β-deficient mouse cells show little dRPase activity (Sobol et al., 2000), but some residual dRP removal by extracts prepared from these cells is still present (Podlutsky et al., 2001). It is possible that while Pol β carries out the bulk of dRP removal from DNA, other activities could be more specifically employed for certain lesions, cell or tissue types, or at certain points of the cell cycle. In *E. coli*, Fpg (formamidopyrimidine-DNA glycosylase) and to a lesser extent endonuclease VIII (Nei) catalyze β-elimination of dRP moiety (Fig. 1). Three mammalian homologues of bacterial Fpg and Nei termed NEIL (Nei-like)-1, -2, and -3 have been identified (Hazra et al., 2002; Hegde et al., 2008). Based on the similarity of their active sites to those of Fpg and Nei, one could expect that they could also display dRPase activity. We have shown that two of these proteins, NEIL1 and NEIL2, are capable of removing dRP lesions from DNA with the efficiency comparable to that of Pol β, and that they can

poly(ADP-ribosyl)ation leading to recruitment of other BER factors.

agents, and its removal is a pivotal step in BER *in vivo* (Sobol et al., 2000).

substitute for Pol β dRPase activity in a reconstituted BER assay (Grin et al., 2006).

dRPase activity can be revealed with 3'-labeled nicked abasic oligonucleotide substrates. Such substrates were prepared by end-filling of a 5'-overhanging oligonucleotide duplexes with 32P-labeled dATP and the consecutive treatment of the duplex with uracil DNA glycosylase (Ung) and APE1. The resulting dRP site is unstable in nucleophilic buffers and is degraded during migration through Tris-containing polyacrylamide gels, therefore the products were stabilized by sodium borohydride reduction immediately after the dRPase-

Fig. 5A illustrates that both NEIL1 and NEIL2 possess a dRP-removing activity. The dRPase activities of NEIL1 and NEIL2 demonstrated the enzyme concentration and time dependence expected of an enzyme-catalyzed reaction (Fig. 5B and data not shown). Notably, the activity of NEIL1 in these experiments appeared higher than that of NEIL1 (Fig. 5B). Both NEIL1 and NEIL2 excised with similar efficiency when A, C, or T were placed opposite the lesion, and the excision of dRP opposite G was 1.5–2-fold lower; Pol β removed dRP equally well from all opposite-base contexts. To confirm that dRP removal by NEIL1 and NEIL2 proceeds by β-elimination, as in Pol β and Fpg, we have performed the reaction in the presence of sodium borohydride, which reduces the Schiff base covalent complexes formed between the catalytic amine nucleophile of dRP lyases and C1' of the dRP site during the reaction (Fig. 5C). Such trapped enzyme–DNA complexes are stable enough to be

**2.3.4 New function of Neil1 and Neil2 as 5′dRP lyase** 

catalyzed reaction.

resolved by regular SDS-PAGE. NEIL1 and NEIL2, as well as Fpg and Pol β, formed lowmobility radioactively labeled complexes.

Fig. 5. **dRPase activity of NEIL1 and NEIL2** (From Grin et al., 2006). (A**)** Cleavage of a dRP moiety by NEIL1 and NEIL2. Lane 1, U-containing oligonucleotide; lane 2, AP-containing oligonucleotide; lanes 3–7, dRP-containing oligonucleotide treated with alkali (lane 4), NEIL1 (lane 6) or NEIL2 (lane 7). In lanes 5–7, the dRP-containing oligonucleotide was stabilized with sodium borohydride to prevent its degradation during electrophoresis. Arrows left to the panels indicate positions of the respective oligonucleotide species after PAGE. (B) Time course of dRP excision by NEIL1 (filled circles) and NEIL2 (open circles). (C) Cross-linking of dRP lyases to a dRP-containing substrate by sodium borohydride: lane 1, no enzyme; lane 2, NEIL1; lane 3, NEIL2; lane 4, Pol β; lane 5, Fpg.

To compare the efficiency of NEIL1 and NEIL2 as dRPases with the same activity of DNA polymerase β, the best-known mammalian dRPase, we have determined steady-state enzyme kinetic constants for all three enzymes. The results of these experiments are summarized in Table 1.


Table 1. Kinetic parameters of the dRPase reaction catalyzed by NEIL1, NEIL2, and DNA polymerase β.

The kinetic data suggest that NEIL1 is as good a dRPase as Pol β, and they both surpassed NEIL2 in their ability to remove dRP from DNA. *K*M of NEIL1 was ~5-fold lower than *K*M of Pol β, indicating that NEIL1 might bind dRP-containing substrate more tightly; on the other hand, Pol β processed the substrate ~5-fold faster than did NEIL1, resulting in nearly equal specificity constants for both enzymes. NEIL2 had an intermediate catalytic constant and the poorest binding of all three dRP lyases compared in this experiment.

New Players in Recognition of Intact and Cleaved AP Sites:

bind to a variety of bulky DNA lesions (Liu et al., 2010; Stros, 2010).

Implication in DNA Repair in Mammalian Cells 317

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

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

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)

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

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,

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

respectively). HMGB1 was also revealed to have weak 5' dRP lyase activity (Fig. 6D).

FEN1 activity. (C) Influence of HMGB1 on APE1 activity. (D) Comparison of the 5'

without a sensitizer and in the presence of 8-methoxypsoralen (100 μM). Protein

repair, nonhomologous end joining. Arrows show the direction of the scan.

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 difference from the repair of U was observed.

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