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

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

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 realized according to the scheme shown in Fig. 2.

New Players in Recognition of Intact and Cleaved AP Sites:

Implication in DNA Repair in Mammalian Cells 313

THF-containing DNA than with control DNA. This suggests that PARP-1 has greater

The next question regarding PARP-1's interaction with AP sites was whether the enzyme is activated for poly(ADP-ribose) synthesis upon binding to the intact AP site. PARP-1 is well known to become activated by binding to DNA strand breaks (Lindahl et al., 1995), and to avoid the presence of confounding DNA ends, we prepared a double-hairpin DNA for use as probe. First, using this hairpin DNA with internal 32P-label, we confirmed the ability of purified PARP-1 to cross-link to the natural AP site. The results showed that double-hairpin DNA bearing the natural AP site was able to cross-link upon NaBH4 reduction, whereas DNA without the AP site (uracil-DNA) failed to yield cross-linked product (data not shown). As expected, THF-containing DNA failed to cross-link. Next, using similar but unlabeled double-hairpin DNA and 32P-NAD+ as substrate for poly(ADP-ribose) synthesis, we examined the activity of PARP-1. Strong PARP-1 auto-modification was observed only in reaction mixtures containing APE1 (data not shown). PARP-1 auto-modification in reaction mixtures with the natural AP site, but without APE1, was weak; this level, however, was more than the background level (data not shown). Under similar conditions, the THF-containing DNA failed to support poly(ADP-ribose) synthesis, but strong synthesis was observed when APE1 was added. These results indicated that PARP-1 interaction with the intact AP site could result in activation, but this activation involved much less auto-

affinity for the THF-containing DNA than for the control DNA (data not shown).

poly(ADP-ribosyl)ation than that observed with APE1-induced strand incision.

to provide poly(ADP-ribose) synthesis activation at the natural AP site.

inhibition of APE1 activity as compared with AP DNA containing single AP site.

Next, to examine PARP-1 auto-modification, purified PARP-1 was first pre-incubated with labeled intact linear AP site-containing DNA. The reaction mixture was then supplemented with NAD+ to allow poly(ADP-ribose) synthesis. Then, the reaction mixture was treated with NaHB4 and analyzed. The results indicated that poly(ADP-ribose) modified enzyme was cross-linked (data not shown). The mechanism of PARP-1 activation was unclear, but presumably involved single strand break formation within the PARP-1 and DNA complex. In light of this result, we were curious to test PARP-1's capacity to conduct strand incision at the AP site via AP lyase activity. As shown in Fig. 4C, PARP-1 was capable of incising AP site-containing DNA, and the activity was similar to that of Pol . In light of PARP-1's AP lyase activity, we also tested for 5'dRP lyase activity. PARP-1 conducted 5'-dRP lyase activity against a pre-incised AP site (Fig. 4C), but the activity was much lower than that of Pol . These results suggested that the endogenous PARP-1 AP lyase activity was sufficient

Interaction of PARP-1 with AP sites appears to be related with regulation of AP site processing. Such a regulation is particularly important for repair of AP sites included in clustered damage, in which chain breaks, oxidized bases and AP sites are grouped within 1– 2 turns of DNA helix and can be situated in both DNA chains. During repair of AP sites within clustered damages additional double strand breaks, which are more cytotoxic, may appear (Yang et al., 2004). PARP-1 influence on hydrolysis of AP sites by APE1 on DNA containing AP site either opposite dAMP or synthetic AP site analogues, was tested (Kutuzov et al., 2011). Along with THF residue, which is most widely used to mimic AP sites, the new AP site analogs were tested (Kutuzov et al., 2011). These analogs were residues of diethylene glycol and decane-1,10-diol. The AP site analogs differ in their sensitivity to the APE1 endonuclease activity. PARP-1 interacts more efficiently with AP sites within clusters that leads to stronger cross-linking with AP sites and more considerable

Results from the MS analyses were searched against a database, and PARP-1 was identified as the first-rank candidate (Mascot probability score of 248, 38% of coverage).

We tested for and found AP site cross-linking by purified PARP-1 (data not shown). Yet, it was not clear whether the cross-linked complex in the extract resulted from PARP-1's reactivity at the intact AP site or a pre-incised AP site.

We next examined purified PARP-1 cross-linking with a linear DNA containing either an intact AP site or pre-incised AP site; in addition to cross-linking probes, these DNAs are substrates for 5'-dRP and AP lyase enzymatic activity (Fig. 4B).

Fig. 4. Interaction of PARP-1 with intact and cleaved AP sites (From Khodyreva et al., 2010b). (A) Cross-linking of mammalian cell extract proteins to circular and linear AP DNA Extracts: whole cell extracts of HeLa, human fibroblasts (HF), MCF-7 and bovine testis nuclear extract (BTNE). (B) Comparison of cross-linking of purified PARP-1 and Pol β with 5'-dRP lyase substrate DNA and AP site-containing DNA. Schematic representations of DNA probes are shown at the top. The \* symbol denotes the position of the 32P-label in the DNA. The bubble-like symbol denotes the presence of the AP site in the DNA. (C) 5' dRP/AP lyase activities of purified PARP-1. The positions of the substrates and products are indicated, and the DNA is illustrated at the bottom.

Cross-linking of PARP-1 was compared with that of Pol β. PARP-1 and Pol β cross-linked to both of these DNA substrates in a concentration-dependent manner. Pol β has a preference for the pre-incised AP site containing-DNA, as compared to the intact AP site. Conversely, PARP-1 does not show a similar preference, yielding similar cross-linking with both probes. The interaction of PARP-1 with the AP site raised the question of the biological relevance of this finding, including whether PARP-1 binds first to the AP site and protects it until repair proteins are recruited. PARP-1 is well known to become activated by binding to DNA strand breaks (Lindahl et al., 1995). Once the AP site became incised by AP endonuclease, PARP-1 became activated and modified by auto-poly(ADP-ribosyl)ation. First, we examined the specificity of PARP-1 interaction with AP site containing-DNA by competition experiments using two types of competitor DNA. A labeled DNA duplex with a 'natural' AP site was used for PARP-1 cross-linking, and the cross-linking was competed either with a control DNA duplex (without an AP site) or a synthetic AP site-containing DNA, tetrahydrofuran (THF), mimicking the AP site. Cross-linking of PARP-1 was reduced with both control and THF-containing DNA. However, the reduction was stronger in the case of

Results from the MS analyses were searched against a database, and PARP-1 was identified

We tested for and found AP site cross-linking by purified PARP-1 (data not shown). Yet, it was not clear whether the cross-linked complex in the extract resulted from PARP-1's

We next examined purified PARP-1 cross-linking with a linear DNA containing either an intact AP site or pre-incised AP site; in addition to cross-linking probes, these DNAs are

Fig. 4. Interaction of PARP-1 with intact and cleaved AP sites (From Khodyreva et al., 2010b). (A) Cross-linking of mammalian cell extract proteins to circular and linear AP DNA Extracts: whole cell extracts of HeLa, human fibroblasts (HF), MCF-7 and bovine testis nuclear extract (BTNE). (B) Comparison of cross-linking of purified PARP-1 and Pol β with 5'-dRP lyase substrate DNA and AP site-containing DNA. Schematic representations of DNA probes are shown at the top. The \* symbol denotes the position of the 32P-label in the DNA. The bubble-like symbol denotes the presence of the AP site in the DNA. (C) 5' dRP/AP lyase activities of purified PARP-1. The positions of the substrates and products

Cross-linking of PARP-1 was compared with that of Pol β. PARP-1 and Pol β cross-linked to both of these DNA substrates in a concentration-dependent manner. Pol β has a preference for the pre-incised AP site containing-DNA, as compared to the intact AP site. Conversely, PARP-1 does not show a similar preference, yielding similar cross-linking with both probes. The interaction of PARP-1 with the AP site raised the question of the biological relevance of this finding, including whether PARP-1 binds first to the AP site and protects it until repair proteins are recruited. PARP-1 is well known to become activated by binding to DNA strand breaks (Lindahl et al., 1995). Once the AP site became incised by AP endonuclease, PARP-1 became activated and modified by auto-poly(ADP-ribosyl)ation. First, we examined the specificity of PARP-1 interaction with AP site containing-DNA by competition experiments using two types of competitor DNA. A labeled DNA duplex with a 'natural' AP site was used for PARP-1 cross-linking, and the cross-linking was competed either with a control DNA duplex (without an AP site) or a synthetic AP site-containing DNA, tetrahydrofuran (THF), mimicking the AP site. Cross-linking of PARP-1 was reduced with both control and THF-containing DNA. However, the reduction was stronger in the case of

as the first-rank candidate (Mascot probability score of 248, 38% of coverage).

reactivity at the intact AP site or a pre-incised AP site.

are indicated, and the DNA is illustrated at the bottom.

substrates for 5'-dRP and AP lyase enzymatic activity (Fig. 4B).

THF-containing DNA than with control DNA. This suggests that PARP-1 has greater affinity for the THF-containing DNA than for the control DNA (data not shown).

The next question regarding PARP-1's interaction with AP sites was whether the enzyme is activated for poly(ADP-ribose) synthesis upon binding to the intact AP site. PARP-1 is well known to become activated by binding to DNA strand breaks (Lindahl et al., 1995), and to avoid the presence of confounding DNA ends, we prepared a double-hairpin DNA for use as probe. First, using this hairpin DNA with internal 32P-label, we confirmed the ability of purified PARP-1 to cross-link to the natural AP site. The results showed that double-hairpin DNA bearing the natural AP site was able to cross-link upon NaBH4 reduction, whereas DNA without the AP site (uracil-DNA) failed to yield cross-linked product (data not shown). As expected, THF-containing DNA failed to cross-link. Next, using similar but unlabeled double-hairpin DNA and 32P-NAD+ as substrate for poly(ADP-ribose) synthesis, we examined the activity of PARP-1. Strong PARP-1 auto-modification was observed only in reaction mixtures containing APE1 (data not shown). PARP-1 auto-modification in reaction mixtures with the natural AP site, but without APE1, was weak; this level, however, was more than the background level (data not shown). Under similar conditions, the THF-containing DNA failed to support poly(ADP-ribose) synthesis, but strong synthesis was observed when APE1 was added. These results indicated that PARP-1 interaction with the intact AP site could result in activation, but this activation involved much less autopoly(ADP-ribosyl)ation than that observed with APE1-induced strand incision.

Next, to examine PARP-1 auto-modification, purified PARP-1 was first pre-incubated with labeled intact linear AP site-containing DNA. The reaction mixture was then supplemented with NAD+ to allow poly(ADP-ribose) synthesis. Then, the reaction mixture was treated with NaHB4 and analyzed. The results indicated that poly(ADP-ribose) modified enzyme was cross-linked (data not shown). The mechanism of PARP-1 activation was unclear, but presumably involved single strand break formation within the PARP-1 and DNA complex. In light of this result, we were curious to test PARP-1's capacity to conduct strand incision at the AP site via AP lyase activity. As shown in Fig. 4C, PARP-1 was capable of incising AP site-containing DNA, and the activity was similar to that of Pol . In light of PARP-1's AP lyase activity, we also tested for 5'dRP lyase activity. PARP-1 conducted 5'-dRP lyase activity against a pre-incised AP site (Fig. 4C), but the activity was much lower than that of Pol . These results suggested that the endogenous PARP-1 AP lyase activity was sufficient to provide poly(ADP-ribose) synthesis activation at the natural AP site.

Interaction of PARP-1 with AP sites appears to be related with regulation of AP site processing. Such a regulation is particularly important for repair of AP sites included in clustered damage, in which chain breaks, oxidized bases and AP sites are grouped within 1– 2 turns of DNA helix and can be situated in both DNA chains. During repair of AP sites within clustered damages additional double strand breaks, which are more cytotoxic, may appear (Yang et al., 2004). PARP-1 influence on hydrolysis of AP sites by APE1 on DNA containing AP site either opposite dAMP or synthetic AP site analogues, was tested (Kutuzov et al., 2011). Along with THF residue, which is most widely used to mimic AP sites, the new AP site analogs were tested (Kutuzov et al., 2011). These analogs were residues of diethylene glycol and decane-1,10-diol. The AP site analogs differ in their sensitivity to the APE1 endonuclease activity. PARP-1 interacts more efficiently with AP sites within clusters that leads to stronger cross-linking with AP sites and more considerable inhibition of APE1 activity as compared with AP DNA containing single AP site.

New Players in Recognition of Intact and Cleaved AP Sites:

mobility radioactively labeled complexes.

Implication in DNA Repair in Mammalian Cells 315

resolved by regular SDS-PAGE. NEIL1 and NEIL2, as well as Fpg and Pol β, formed low-

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

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

NEIL1 0.21±0.03 0.65±0.04 3.1 NEIL2 2.2±0.7 1.6±0.1 0.74 Pol β 1.0±0.1 3.0±0.1 3.0

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

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

*K*M, μM *k*cat, min–1 *k*cat/*K*M, μM–1·min–1

1, no enzyme; lane 2, NEIL1; lane 3, NEIL2; lane 4, Pol β; lane 5, Fpg.

poorest binding of all three dRP lyases compared in this experiment.

summarized in Table 1.

polymerase β.

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