3′-Phosphodiesterase activity of human apurinic/apyrimidinic endonuclease at DNA double-strand break ends

Dongchul Suh 1 David M. Wilson 1 III 0 1 2 Lawrence F. Povirk 1 0 Department of Molecular and Cellular Toxicology, Harvard University School of Public Health , Boston, MA 02115, USA 1 Department of Pharmacology and Toxicology, Medical College of Virginia, Virginia Commonwealth University , Richmond, VA 23298, USA 2 Present address: Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory , Livemore, CA 94551, USA *To whom correspondence should be addressed. Tel: +1 804 828 9640; Fax: +1 804 828 8079; Email: - In order to assess the possible role of human apurinic/ apyrimidinic endonuclease (Ape) in double-strand break repair, the substrate specificity of this enzyme was investigated using short DNA duplexes and partial duplexes, each having a single 3-phosphoglycolate terminus. Phosphoglycolate removal by Ape was detected as a shift in mobility of 5-end-labeled DNA strands on polyacrylamide sequencing gels, and was quantified by phosphorimaging. Recombinant Ape efficiently removed phosphoglycolates from the 3-terminus of an internal 1 base gap in a 38mer duplex, but acted more slowly on 3-phosphoglycolates at a 19 base-recessed 3-terminus, at an internal nick with no missing bases, and at a double-strand break end with either blunt or 2 base-recessed 3-termini. There was no detectable activity of Ape toward 3-phosphoglycolates on 1 or 2 base protruding single-stranded 3-overhangs. The results suggest that both a singlebase internal gap, and duplex DNA on each side of the gap are important binding/recognition determinants for Ape. While Ape may play a role in repair of terminally blocked double-strand breaks, there must also be additional factors involved in removal of at least some damaged 3-termini, particularly those on 3-overhangs. Oxidative damage to DNA in living cells is induced by ionizing radiation (1), by reactive oxygen species produced by aerobic metabolism (2,3), and by some antitumor agents such as neocarzinostatin and bleomycin (4). Repair of oxidative damage to both the bases and the backbone of DNA requires several distinct repair pathways incorporating numerous enzymatic activities (46). Oxidative damage to the sugar moiety of DNA often results in DNA breaks with damaged ends, and removal of terminal 3-blocking groups is an essential early step in repair of these lesions. However, while several apurinic/apyrimidinic (AP) endonucleases have been shown to remove 3-phosphoglycolates (PGs) from single-strand break termini (5,7), their activity toward 3-blocks on double-strand break ends has not been carefully examined. Ape, also called Hap1 (9), is the major AP endonuclease of human cells (5,7,8). In order to determine whether Ape can remove 3-blocks from double-strand break ends, a series of defined DNA substrates bearing PGs at blunt, recessed and overhanging 3-termini were designed and constructed. In addition to assessing its possible role in double-strand break repair, the results help to define crucial factors involved in substrate recognition by Ape. MATERIALS AND METHODS Materials Bleomycin was obtained from Sigma (St. Louis, MO), and was dissolved in distilled water at a concentration of 2 mM as determined from the A295 (e 295 = 14 100) (10). Fe(III)Bleomycin was prepared by adding an equal amount of 2 mM ferric ammonium sulfate (dissolved in 0.5 mM HCl to prevent ferric hydroxide precipitation), and was stored at 20 C. Ferric ammonium sulfate, magnesium chloride, potassium chloride, boric acid, Tris base, and ammonium peroxydisulfate were purchased from Sigma Chemical Company (St. Louis, MO). Urea, acrylamide, and bis-acrylamide were obtained from Fisher Scientific Co. (Fair Lawn, NJ). Depurinated pUC19 plasmid was prepared by heat treatment of the plasmid at pH 4 as described (11). Preparation of DNA substrates A 25mer oligonucleotide (Fig. 1) was phosphorylated at the 5 end with [g -32P]ATP (5500 Ci/mmol, Du Pont Chemical Co., Wilmington, DE) and subsequently with an excess of unlabeled ATP using T4 polynucleotide kinase according to standard procedures (12). The radiolabeled 25mer was annealed to the complementary 10mer oligonucleotide by incubating at 70 C for 10 min followed by slow cooling, over 2 h, to 15 C. Reaction mixtures for bleomycin-induced cleavage contained 25 mM HEPES-NaOH, pH 7.5, 2 m M 5-32P-end-labeled duplex, 0100 m M Fe(III)bleomycin, and 1 mM H2O2. Bleomycin was added last, and the mixture was incubated at 0 C for 1 h. After ethanol precipitation, DNA samples were mixed with an equal volume of formamide containing 20 mM EDTA, and then subjected to electrophoresis on a 24% polyacrylamide denaturing gel. The band corresponding to the 3-PG-terminated 19mer was located by autoradiography, cut from the gel and eluted in 1 mM EDTA. The eluate was filtered (0.2 m m Gelman Acrodisc) and evaporated to a volume of 1 ml, and the 19mer was further purified by reverse-phase HPLC (13). Using the same slow cooling procedure, the purified 3-PG 19mer and a 5-phosphorylated 18mer were annealed to a complementary 38mer, as shown in the last step in Figure 1. Other DNA substrates were constructed in a similar manner by annealing the 5-phosphorylated 19mer to oligomers of different lengths, to give structures with 3-blocked ends in various configurations, as shown in Figure 2. To obtain an oligomeric substrate with an AP site, a 38mer duplex containing a single uracil (5-end-labeled GGGACTCTCGAGGAATGCGUCAGCTAATGGCTAGCGGC annealed to a complementary 38mer with G opposite the U) was treated with 2 U uracil DNA glycosylase (14) (provided by D. W. Mosbaugh, Oregon State University) for 20 min at 37 C, and placed on ice until treatment with Ape. Enzymatic assays of Ape Recombinant human AP endonuclease (Ape) was isolated as a glutathione S-transferase fusion protein following biosynthesis in an overproducing Escherichia coli strain. Following cleavage by factor Xa, the clipped Ape protein was purified to apparent homogeneity as described in detail previously (15). This preparation gave a single band on silver-stained polyacrylamide gels and typically had a specific AP endonuclease activity of 500750 U/m g, where one unit is defined as the amount of enzyme required to cleave one pmol of AP sites per min at 37 C under standard assay conditions (16). This specific activity is comparable to that of highly purified native Ape from HeLa cells (15). Each reaction mixture contained 50 mM HEPES-NaOH, pH 7.5, 50 mM KCl, 100 m g/ml bovine serum albumin, 10 mM MgCl2 and DNA substrate in a volume of 25 m l. Following enzyme addition, the reaction mixtures were incubated for 10 min at 37 C and then the reaction was stopped by addition of 0.1 M EDTA followed by immersion in an ice bath. Polyacrylamide gel electrophoresis Following Ape treatment, a 5 m l aliquot of each sample was added to 5 m l formamide, loaded onto a 24% polyacrylamide sequencing gel (30 40 0.8 cm) and electrophoresed for 6 h at 50 W in a buffer composed (...truncated)