PAGE 5 OF 10 Nucleic Acids Research, 2007, Vol. 35, No. 17 e112 Figure 2. Schematic representation of plasmid and oligonucleotide DNA substrates. (A) Sequence of the uracil-containing fragment of pUN1 and restriction sites are shown. After the repair reaction, DNA products were restricted with the indicated enzymes, and fragments were separated by 20% denaturing PAGE. DNA fragment sizes and descriptions of DNA synthesis products are indicated. (B) The sequence of 55-bp oligonucleotide containing uracil at position 32 and a strategic restriction site is shown. The sequence of the oligonucleotide substrate is identical to the 41-mer fragment of plasmid in (A). DNA fragment sizes and descriptions of DNA synthesis products are indicated. intermediate was observed in some cases, and again, this material was considered as background. The XhoI digestion also yielded a 23-mer fragment containing the LP BER incorporations (Figure 2B). Typical results obtained with a laboratory reference tissue extract, BTNE, and the plasmid and oligonucleotide substrates described above are shown in Figure 3. Repair of the uracil-DNA plasmid substrate and digestion with KpnI resulted in the labeled 41-mer fragment; very little stalled 25-mer intermediate was observed (Figure 3A). In contrast, with the THF-containing plasmid substrate, a signiﬁcant band representing the stalled intermediate was observed with KpnI digestion alone (Figure 3A). This indicated accumulation of the intermediate at the step just prior to strand displacement synthesis in LP BER. The results obtained with the oligonucleotide substrates without restriction enzyme digestion were similar, except the stalled intermediate was more abun- dant (Figure 3B). Double digestion of the plasmid products yielded the 25-mer and 16-mer fragments described above (Figure 3C). The results illustrated that most of the uracil-DNA repair corresponded to SN BER, as expected from earlier results (9). Analysis of the product formed on the THF-containing oligonucleotide substrate revealed that the amount of stalled intermediate was greater than the LP BER product, and with uracil-DNA the results were similar to those with plasmid substrate (Figure 3D). Comparison of steady-state kinetic features of repair with plasmid and oligonucleotide substrates The main goal of this study was to compare kinetic features of BER product formation with plasmid and oligonucleotide substrates. We measured the initial rates of BTNE-mediated BER for the two forms of substrate under conditions of substrate excess. The rates of product formation with various uracil-DNA substrate concentra- tions were linear over the ﬁrst 10 min of incubation, as illustrated at one substrate concentration in the experi- ment shown in Figure 4. Kinetic features for BTNE- mediated BER with the two substrates are summarized in Table 1. The maximal velocities (Vmax) of BER product formation for the plasmid and oligonucleotide substrates were similar, but the Km for the plasmid substrate, pUN1, was slightly higher than that for the oligonucleotide substrate. The resultant eﬃciency (Vmax/Km) was slightly higher (3-fold) with the oligonucleotide substrate. These results indicate that similar rates of uracil-DNA repair can be obtained with the two forms of substrate, especially when they are used at concentrations high enough to support near maximal synthesis, i.e. Km.