e112 Nucleic Acids Research, 2007, Vol. 35, No. 17 PAGE 6 OF 10 Figure 3. Comparison of BER reaction products using plasmid and oligonucleotide substrates. In order to compare the BER reaction products using plasmid-based or oligonucleotide-based assay, the repair reaction was performed with lesion-speciﬁc DNA substrate, as indicted at the top of each panel, and BTNE. The reaction conditions and product analyses were as described under ‘Materials and Methods’ section. Panels (A–D), BER reaction was performed in a 10 ml reaction mixture that contained 50 nM plasmid/oligonucleotide DNA (uracil- or THF-containing DNA) and 10 mg BTNE. Incubation was for 30 min at 378C. The reaction products were puriﬁed, digested with the indicated restriction enzyme (s), and then separated by 20% denaturing PAGE. Panels (A) and (B) show the total BER products represented by 41-mer and 55-mer DNA fragments obtained with uracil or THF-containing plasmid, pUN1 or pUN2, and oligonucleotide substrates, respectively. The 25-mer and 32-mer DNA fragments observed with plasmid and oligonucleotide substrates, respectively, represented unligated or stalled intermediates. Panels (C) and (D), the remaining reaction products in (A) and (B) were digested with KpnI or KpnI plus XhoI (Panel C), and XhoI (Panel D), respectively. This resulted in 25-mer (SN BER) and 16-mer (LP BER) DNA fragments from plasmid substrate and 32-mer (SN BER) and 23-mer (LP BER) DNA fragments from oligonucleotide substrate, respectively. In cases where unligated or stalled intermediates were observed (i.e. the 25-mer and 32-mer fragments), the counts in these molecules were considered as background and subtracted for the calculation of SN BER. The positions of total BER, SN BER/stalled and LP BER products are indicated. Use of plasmid substrate in measurement of the ratio of SN to LP BER In some cases, it is desirable to measure the relative amounts of the SN and LP BER subpathways. Adjusting the sequence of the repair patch to eliminate ambiguity from incorporation of more than one labeled dNMP in each subpathway can facilitate this measurement. To accomplish this type of measurement, we constructed an alternate plasmid substrate termed ‘pPAL1’ (Figure 5A). This plasmid was designed for use in BER assays along with [a-32P]dCTP as the labeled nucleotide substrate. To illustrate use of this method, incubations were conducted with MEF extract. Double digestion of the reaction product with NcoI and SacI yielded a 47-mer representing the entire excision repair patch and a small amount of stalled intermediate (Figure 5B). Digestion with these enzymes plus KpnI yielded a 22-mer with the ﬁrst dNMP incorporated, labeled dCMP, and a 25-mer with labeled dCMP corresponding to the second nucleo- tide incorporated and potentially the 25th dCMP incor- porated into the repair patch (Figure 5A). Since the repair patch in LP BER is shorter than 25 residues, potential dCMP incorporation at the 25th position can be ignored. Therefore, quantiﬁcation of [a-32P]dCMP incorporated into the 22-mer and 25-mer fragments, respectively, yields the molar amounts of total BER product (SN and LP BER) and LP BER product, as illustrated in Figure 5. In this case, the ratio of SN BER to LP BER was approximately 60:40 (Figure 5C), as expected from earlier results obtained by diﬀerent methods (21,37). Repair patch analysis for long patch base excision repair products As noted in Figure 2, the plasmid and oligonucleotide substrates can be used for repair patch size assessment of LP BER reaction products. Such analysis involves restriction enzyme digestion and then the electrophoretic separation and quantiﬁcation of various labeled fragments (32P-labeled dNMP incorporation). Typical results from this type of analysis are illustrated in Figure 6. In the experiment shown, we used the THF-containing plasmid, pUN2, (Figure 1) so that repair corresponded to the LP BER subpathway only, along with an extract known to be proﬁcient in LP BER (i.e. from MEFs). Measurement of dCMP and dTMP incorporated in separate incubations was conducted (Figure 6). In this case for LP BER, the amount of dCMP incorporation into the ﬁrst dNMP position (i.e. 25-mer) is equal to the amount of dTMP incorporation into the second dNMP position. Therefore, to evaluate LP BER synthesis beyond the second