PAGE 3 OF 10 step described above. The resulting plasmids containing either uracil or THF were designated as pUN1 or pUN2, respectively. Routinely, the ratio of lesion-speciﬁc plasmid to starting plasmid was greater than 3 : 1 in the experi- ments shown here. Preparation of lesion-specific oligonucleotide DNA substrates Unphosphorylated 55-mer oligonucleotide (100 mM) with 30 inverted dT (50-TCGGTACCCGGGGATCGAGT CGAATGCATGCCTCGAGTCTAGAGGTACCAGAT CT-30) containing either uracil or THF at position 32 (underscored C) was annealed to its complementary unphosphorylated 55-mer in 10 mM Tris–HCl, pH 8.0 and 100 mM NaCl, and then the DNA solution was diluted to 1 mM in the same buﬀer. The solution was divided into aliquots and stored at 808C. Preparation of lesion-specific pPAL1 plasmid The plasmid pGL4.10TK Kpn (5.0 kb) was constructed from pGL4.10 (Promega, Madison, WI, USA) by insert- ing the TK promoter between KpnI and HindIII sites. During this process the KpnI site was lost. For the in vitro BER plasmid assay, pPAL1 was prepared by replacing the HindIII-ApaI fragment of pGL4.10TK Kpn with an oligonucleotide that contained a uracil at a new KpnI site and unique NcoI and SacI restriction sites. To prepare the HindIII-ApaI fragment, pGL4.10TK Kpn was digested with ApaI and subsequently treated with calf intestine alkaline phosphatase. The resultant plasmid was digested by HindIII and puriﬁed by 1% agarose gel electrophoresis. Then a 73/81-bp hybrid oligonucleotide was prepared by annealing unphosphory- lated 73-mer 50CTTCTTAATGTTTTTGGCATCTTCC ATGGTGGCTTTACCAAGGTAUCAGTAAGTATTA ATTAAGGAGAGCTCA-30 and 50-phosphorylated 81-mer 50-AGCTTGAGCTCTCCTTAATTAATACTT ACTGGTACCTTGGTAAAGCCACCATGGAAGATG CCAAAAACATTAAGAAGGGCC-30 DNA. Ligation of this oligonucleotide into the HindIII-ApaI digestion product of pGL4.10TK Kpn was carried out as described (35). The closed circular DNA was separated by 1% low-melting temperature agarose gel electrophor- esis containing SYBR Gold nucleic acid gel stain (Invitrogen), and the plasmid was recovered using a gel extraction kit (Qiagen). The resulting plasmid, pPAL1, was obtained in microgram quantities. Nuclear extract preparation Nuclear extract was prepared from bovine testis essen- tially as described previously (36). Brieﬂy, 100 g of bovine testis was minced in 300 ml buﬀer A [10 mM Hepes, pH 8.0, 1.5 mM MgCl2, 10 mM NaCl, 0.5 mM dithio- threitol (DTT), 10 mM sodium metabisulﬁte, 0.1 mM 4-(2-aminoethyl)benzenesulfonylﬂuoride (AEBSF), 1 mM benzamidine, 1 mg/ml leupeptin and 1 mg/ml pepstatin A] and homogenized using four 10 s bursts with a blender at 48C. The homogenate was pelleted by centrifugation at 10 000 g for 10 min at 48C. Pellet fraction was resuspended in 150 ml of buﬀer B (same as buﬀer A, except it contained 1 M NaCl) and blended using three Nucleic Acids Research, 2007, Vol. 35, No. 17 e112 5 s bursts. The homogenate was centrifuged at 100 000 g for 1 h at 48C. The clear supernatant fraction was brought to 40% saturation by adding solid ammonium sulfate slowly with stirring; stirring was continued for 1 h at 48C. The precipitate was recovered by centrifugation at 14 000 g for 20 min at 48C. The pellet fraction was resuspended in 50 ml of buﬀer C (25 mM Hepes, pH 7.5, 100 mM NaCl, 1 mM DTT, 10 mM sodium metabisulﬁte, 0.5 mM EDTA, 0.1 mM AEBSF, 1 mM benzamidine, 1 mg/ml leupeptin, 1 mg/ml pepstatin A and 20% glycerol) and dialyzed against the same buﬀer. The dialyzed nuclear extract was clariﬁed by centrifugation at 10 000 g for 20 min. The clear supernatant fraction was referred to as the bovine testis nuclear extract (BTNE) and stored at 808C. Protein concentration was determined with a Bio-Rad protein assay kit using bovine serum albumin (BSA) as a standard. Cell extract preparation Whole cell extract was prepared as previously described (4). Brieﬂy, cells were washed twice with phosphate-buﬀered saline at room temperature, detached by scraping, pelleted by centrifugation and resuspended in Buﬀer I (10 mM Tris–HCl, pH 7.8, 200 mM KCl and protease inhibitor cocktail). An equal volume of Buﬀer II (10 mM Tris–HCl, pH 7.8, 200 mM KCl, 2 mM EDTA, 40% glycerol, 0.2% Nonidet P-40, 2 mM DTT and protease inhibitor cocktail) was added. The suspension was rotated for 1 h at 48C, and the resulting extract was clariﬁed by centrifugation at 14 000 rpm at 48C. The protein concentration of the extract was determined as above, and aliquots were stored at 808C. In vitro BER assay Uracil- and THF-containing plasmids were designed as described above to quantify total BER, SN BER and LP BER in the same reaction. A 48-nt N.BstNB1 fragment containing uracil or THF (Figure 1) was inserted in pUC19, which upon digestion with strategic restriction enzymes, as indicated in Figure 2, would generate frag- ments representing total BER product (41-bp KpnI fragment), SN BER plus LP BER (25-bp KpnI-XhoI fragment) and LP BER (16-bp XhoI-KpnI fragment). BER assays were performed using reaction mixtures of 10 ml ﬁnal volume that contained 20–500 nM plasmid or oligonucleotide DNA substrate, 10 mg of extract, 50 mM Tris–HCl, pH 7.5, 5 mM MgCl2, 20 mM NaCl, 1 mM DTT, 4 mM ATP, 20 mM each of dATP, dGTP and dTTP or dCTP and 2.3 mM [a-32P]dCTP or [a-32P]dTTP, as indicated. The incubation was at 378C for 1 to 30 min, as indicated. Reactions were terminated by adding 1 ml of 0.5 M EDTA and 90 ml TE (10 mM Tris– HCl, pH 8.0 and 1 mM EDTA), and this was followed by phenol/chloroform extraction and ethanol precipita- tion. To facilitate ethanol precipitation, 100 ng of carrier tRNA was added to each reaction mixture. The resulting DNA pellet fraction was dissolved in H2O and divided into three equal portions. These DNA-containing portions were subjected to restriction enzyme digestion as follows: (i) no enzyme, (ii) KpnI and (iii) KpnI plus