e112 Nucleic Acids Research, 2007, Vol. 35, No. 17 XhoI. The reaction mixtures were incubated for 90 min at 378C. The resulting reaction products were separated by 20% denaturing polyacrylamide gel electrophoresis (PAGE). The gels were dried, scanned with a Typhoon PhosphorImager and the data were analyzed with ImageQuant software. The BER assay, with uracil- containing plasmid, pPAL1, also was performed under similar reaction conditions as above, and the repaired DNA was restricted with NcoI/SacI and/or KpnI, as indicated in Figure 5. The experiments were repeated several times, and representative images and average values of SN BER and LP BER are shown. In vitro BER assays using the 55-bp oligonucleotide duplex DNA and cellular extracts were performed under similar reaction conditions as with the plasmid DNA. RESULTS Preparation of plasmid DNA substrates A plasmid DNA substrate containing either uracil or THF opposite guanine was prepared as outlined in Figure 1. Uracil opposite guanine was chosen because this mis- match is a widely used initiating lesion for studies of in vitro BER. The THF abasic site lesion was chosen as a reference because all BER with this lesion occurs through the LP BER subpathway, since DNA ligation after the gap-ﬁlling step in BER is blocked by the persistent THF group (12,13). A key feature in plasmid preparation was the use of the single-strand cutting restriction enzyme N.BstNB1, such that a single-strand gap of 48 nt was introduced into the plasmid and the released 48-mer was removed (Figure 1, see penultimate step). Eventually, a synthetic 48-mer uracil-containing or THF-containing oligonucleotide was annealed into the gap, and the nicks were ligated with good yield (Figure 1, ﬁnal step). The plasmids (pUN1 or pUN2) thus prepared could be puriﬁed in milligram quantities, enabling the use of as much as micromolar ﬁnal concentrations of plasmid substrate in BER reaction mixtures. In addition, these plasmids were designed such that the lesion was ﬂanked with two KpnI sites (Figure 2A). Digestion of BER reaction products with this enzyme yielded a 41-nt fragment (41-mer). Upon denaturing gel electrophoresis, the incorporation of labeled dNMPs into the repair patch within this fragment could be measured. Quantitative assessment of the repair product formed during the BER incubation could be obtained by double digestion with KpnI and XhoI and use of 32P-labeled dCTP as one of the nucleotide substrates. In this way, incorporation of labeled dCMP into the ﬁrst dNMP position of the repair patch was used to quantify the molar amount of repair product formed. This digestion also produced a 16-mer fragment corresponding to the second through seventeenth incorporations by the LP BER sub- pathway, and should contain most of the LP BER product (Figure 2A). In some cases, KpnI digestion alone yielded a 25-nt (25-mer) labeled fragment reﬂecting accumulation of stalled/unligated BER intermediate, i.e. intermediate prior to the DNA ligation step in SN BER or the strand displacement step in LP BER. In the experiments shown PAGE 4 OF 10 Figure 1. Construction of lesion-speciﬁc plasmid substrates. Lesion- speciﬁc plasmid substrates for in vitro BER assays, pUN1 and pUN2, were constructed as described under ‘Materials and Methods section’. The pUC19 was cut with BamHI and a 43-bp fragment was inserted to create pUC19N that contained two N.BstNB1 sites. Then, the pUC19N was digested with N.BstNB1 to generate a 48-nt gap. The 48-nt fragment was removed by hybridization to a biotinylated-tagged comple- mentary oligonucleotide and followed by adsorption onto streptavidin- coated magnetic beads. Next, the plasmid, pUN1 or pUN2, was created by re-hybridization and ligation of gapped pUC19N to the 50-phosphorylated 48-mer oligonucleotide containing either uracil or THF, respectively, as shown. subsequently, this material was considered as background since it did not represent complete repair. Digestion with other restriction enzymes is also illustrated in Figure 2. Two fragments are produced by double digestion with XhoI and XbaI, a 6-mer representing the second through seventh incorporations in the repair patch and a 23-mer representing incorporations out to the 30th position in the repair patch (Figure 2A). With the oligonucleotide substrates, a related strategy was used for analysis of BER reaction products. These oligonucleotide substrates had lesions and sequences identical to those in the plasmid substrates (Figure 2). In the case of products formed in reaction mixtures with labeled dCTP, digestion with XhoI yielded a 32-mer containing the ﬁrst labeled dCMP incorporated (Figure 2B), allowing assessment of the molar amount of product formed. The stalled or unligated BER