The capacity of human poly(ADP-ribose) polymerase-1 (PARP-1) to interact with intact apurinic/apyrimidinic (AP) sites in DNA has been demonstrated. In cell extracts, sodium borohydride reduction of the PARP-1/AP site DNA complex resulted in covalent crosslinking of PARP-1 to DNA; the identity of cross-linked PARP-1 was confirmed by mass spectrometry. Using purified human PARP-1, the specificity of PARP-1 binding to AP site-containing DNA was confirmed in competition binding experiments. PARP-1 was only weakly activated to conduct poly(ADP-ribose) synthesis upon binding to AP site-containing DNA, but was strongly activated for poly (ADP-ribose) synthesis upon strand incision by AP endonuclease 1 (APE1). By virtue of its binding to AP sites, PARP-1 could be poised for its role in base excision repair, pending DNA strand incision by APE1 or the 5′-dRP/AP lyase activity in PARP-1.Schiff base | apurinic/apyrimidinic site-binding protein T he apurinic/apyrimidinic (AP) site is considered to be a common lesion in genomic DNA, arising at a frequency of 10,000 to 50,000 lesions per mammalian cell per day (1). If unrepaired, AP sites present mutagenic and cytotoxic consequences to the cell (2). The loss of DNA bases and attendant formation of AP sites in DNA occurs spontaneously as a result of hydrolytic cleavage of N-glycosylic bonds. AP sites are also generated through glycosylase-catalyzed removal of damaged bases during the early stage of base excision repair (BER) (3). The number of AP sites can increase dramatically under stressful conditions such as X-ray or UV light irradiation and alkylating agent exposure (4). AP sites in isolated DNA are stable enough for their use as substrates in enzymatic assays, but AP sites can be converted to single-strand breaks by alkali treatment, heating, or nucleophilic attack at the aldehydic C1′ group (5). Intact AP sites in vivo can be stable enough to be mutagenic (6) during replication. Measurements of steady-state levels of AP sites in mammalian cells have varied somewhat, but are in the range of approximately 1 site per 10 6 nucleotides (4, 7). Nevertheless, repair of AP sites is thought to be extremely rapid, thus minimizing the steady-state AP site level (8). In mammalian cells, the repair of AP sites is generally initiated through strand incision by AP endonuclease 1 (APE1), the second enzyme of the canonical BER pathway (9, 10). AP sites also can be incised through β-elimination via the activity of DNA glycosylases and other enzymes with associated AP lyase activity (3,11,12). In this case, strand incision forms a singlenucleotide gap with the AP site sugar phosphate at the 3′ margin and phosphate at the 5′ margin, and this intermediate is further processed by polynucleotide kinase, APE1, or other BER enzymes (12).During AP site cleavage by the AP lyase activity of DNA glycosylases, an aldimine intermediate between the C1′ atom of deoxyribose and a primary amine group in the protein is formed (3,13,14). This intermediate, termed the Schiff base, can be reduced in vitro by ...

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