PAGE 7 OF 10 Nucleic Acids Research, 2007, Vol. 35, No. 17 e112 Figure 4. Steady-state kinetic analysis of the repair reaction using plasmid and oligonucleotide substrates. The reaction conditions and products analyses were as described under ‘Materials and Methods section’. Repair reaction was performed under conditions of substrate excess with BTNE using either uracil-containing plasmid (open circle) or uracil-containing oligonucleotide (closed circle) substrate. The concentrations of plasmid and oligonucleotide substrates were 450 nM and 200 nM, respectively. Aliquots were taken at the indicated time intervals, and the DNA products were analyzed as in Figure 3. Time courses of product formation for the plasmid and oligonucleotide substrates are shown. BER products were quantiﬁed with ImageQuant software, and the data were ﬁtted to a straight-line equation. The initial rates of BTNE-mediated BER for the plasmid and oligonucleotide substrates were 0.67 and 1.85 fmol dCMP incorporated/min, respec- tively. The experiments were repeated three times, and a representative graph from one experiment is shown. nucleotide, the amount of dTMP incorporated into the second position of the 16-mer could be subtracted from total dTMP incorporation into the 16-mer; this synthesis in the 16-mer for dTMP incorporation was similar to that for dCMP incorporation (Figure 6B and C). Further analysis of repair patch size is illustrated in Figure 7. Typical results (Figure 7A) and the strategy for analysis using 32P-labeled dCMP incorporation (Figure 7B) were as follows: Restriction with KpnI yielded a 41-nt fragment (41-mer) representing repair patch synthesis, i.e. from one up to 17 nucleotide incorporations (Figure 7A, lane 1, and B). With KpnI digestion, some 25-mer BER intermediate was also observed, reﬂecting stalling at this intermediate. Incorporation of the ﬁrst nucleotide in the repair patch, dCMP, was measured after double digestion with KpnI and XhoI, yielding the 25-mer and the 16-mer (Figure 7A lane 2). The 16-mer fragment reﬂected three LP BER dCMP incorporations (Figure 7A , lane 2, and B). For LP BER synthesis at the 2nd through 7th nucleotides and longer repair patches, respectively, we measured dCMP incorporation into the corresponding fragments after double digestions with XhoI and XbaI, i.e. two fragments (6-mer and 23-mer) as illustrated in Figure 7 (Figure 7A, lane 3, and B). Incorporation into the 6-mer was greater than that into the 23-mer, indicating that most of the LP BER corresponded to repair patches of 7 nt or less (Figure 7C). Figure 5. Assessment of the ratio of SN to LP BER after repair incubation. (A) Schematic diagram of uracil-containing plasmid, ‘pPAL1’, is shown. A uracil residue (U) was placed in a central position of the NcoI/SacI fragment. Three strategic restriction enzyme sites, NcoI, KpnI and SacI, were designed around the lesion such that SN and LP BER could be analyzed in the same repair reaction mixture. After completion of the BER reaction in the presence of [a-32P]dCTP, repair products were restricted with either NcoI and SacI or NcoI, SacI and KpnI, which generated 47-, 25- and 22-mer DNA fragments representing total, LP and SN BER products, respectively. To calculate SN and LP BER products, counts in the 25-mer fragment were subtracted from the total counts in 22-mer fragment, because for every incorporation of dCMP at the second position, next to uracil, one dCMP will be incorporated ﬁrst at the lesion site. (B) pPAL1 (20 nM) was incubated with 10 mg of MEF extract in 50 mM Tris–HCl, pH 7.5, 5 mM MgCl2, 20 mM NaCl and 1 mM DTT. The reaction was conducted at 378C for 30 min in the presence of 20 mM each of dATP, dGTP, dTTP and 2.3 mM [a-32P]dCTP. A 16-mer radiolabeled DNA fragment was added in each reaction mixture as an internal control prior to phenol/chloroform extraction and ethanol precipita- tion. The reaction products were analyzed by 15% denaturing PAGE. The combinations of restriction enzymes used are shown at top of the PhosphorImager panel. The description of each radiolabeled band is indicated on the right-hand side of the image. (C) Quantiﬁcation of total, SN and LP BER is shown in a bar graph. Band intensity of each radiolabeled DNA fragment, 47-, 25- and 22-mer, was measured in terms of arbitrary PhosphorImager units and plotted as total BER, SN BER and LP BER. The experiments were repeated three times and a PhosphorImage of a representative experiment is shown. DISCUSSION We made use of a recently acquired method for obtaining large amounts of plasmid DNA after a preparative in vitro ligation step (30). A plasmid with a single-strand 48 base gap was prepared in high yield and in pure enough form to enable annealing a 48-mer lesion-containing oligonucleo- tide into the gap. After ligation and puriﬁcation of the