e112 Nucleic Acids Research, 2007, Vol. 35, No. 17 lesion-containing plasmid DNA substrate, a relatively large amount of DNA was available for use in routine BER incubations (Figure 1). The availability of such large quantities of plasmid substrate made it possible for us to compare steady-state kinetic values for BER obtained with an oligonucleotide substrate with those obtained with a plasmid substrate. Such a comparison had been Table 1. Steady-state kinetic measurements of BER using plasmid and oligonucleotide substrates Oligonucleotide Plasmid Km (nM) Vmax Vmax/Km (nM/min) (min 1 51.4 (9.9) 0.05 (0.02) 9.7 (4.3) Km Vmax Vmax/Km 10 4) (nM) (nM/min) (min 1 10 4) 216 (27.3) 0.09 (0.03) 4.0 (1.0) Mean ( SE) of four independent experiments. PAGE 8 OF 10 unresolved and remained an important consideration. Some investigators maintained that plasmid substrates are more biologically relevant or useful than oligonucleotide substrates. On the other hand, others have held the opposite viewpoint asserting that plasmid substrates are of limited and specialized use because of the necessity to employ miniscule concentrations of substrate DNA and long incubation periods, both of which potentially confound interpretations on BER capacity measurements. Our results indicated that the two forms of substrate yielded similar values for rates of repair in a nuclear extract, provided the DNA substrate concentration was high enough in the reaction mixture. A precaution emerged from the observation that Km for plasmid substrate was higher than Km for oligonucleotide substrate. An explanation for this diﬀerence was not examined. However, on a practical level, when a substrate excess or steady-state approach is used to quantify Figure 6. Incorporation of labeled dCMP and dTMP into the repair patch produced during LP BER of the THF-containing plasmid. The repair reaction was performed with THF-containing plasmid, pUN2 and MEF extract. The reaction conditions and product analyses were as described under ‘Materials and Methods section’. (A) BER reaction was performed in a10 ml reaction mixture that contained 20 nM pUN2, 10 mg MEF extract and either 32P-dCTP (lanes 1and 2) or 32P-dTTP (lanes 3 and 4). Incubation was for 30 min at 378C. The reaction products were analyzed as in Figure 3. The restriction enzymes used are shown at the top of the PhosphorImager panel. The description of each radiolabeled band is indicated on both sides of the image. (B) Quantiﬁcation of total BER (41-mer), SN BER (25-mer) and LP BER (16-mer) is shown in a bar graph. A small portion of each reaction was spotted on the gel ﬁlter for calculations of incorporation of 32P-dCMP or 32P-dTMP in DNA. The band intensity of each radiolabeled DNA fragment, 41-, 25- and 16-mer, was measured in terms of arbitrary PhosphorImager units and then converted into relative dCMP or dTMP incorporation. The experiments were repeated three times, and the PhosphorImage of a representative experiment is shown. (C) The positions of dCMP (ﬁlled circle) or dTMP (cross) incorporation in the 41-, 25- and 16-mer fragments, and the restriction sites are indicated.