Figure 5. Analysis of SN- and LP-BER capacities using a uracil-containing oligonucleotide duplex DNA substrate.

Incorporation of [α-³²P]dCMP (*C) was measured in the presence of ddTTP (ddT) to discriminate between SN-BER and LP-BER. (A) Schematic representation of the substrate DNA and predicted BER reaction products and intermediates. The sizes and intermediates were 1-nt addition, SN-BER (15-bp); 2-nt addition, LP-BER (16-bp); and complete BER product (ligated SN-BER [35-bp]). A 35-bp oligonucleotide containing a uracil residue at position 15 was utilized in the BER assay. In SN-BER, [α-³²P]dCMP was incorporated in place of uracil and directly ligated to complete the repair. In LP-BER, ddTMP was incorporated following incorporation of [α-³²P]dCMP in place of uracil, and the resulting dideoxy-terminated intermediate prevented subsequent primer extension DNA synthesis, as well as the ligation reaction. The asterisks designate the position of the radiolabeled CMP group. (B and C) Photographs of PhosphorImager image illustrating LP-BER analysis. A 35-bp duplex oligonucleotide containing a uracil residue at position 15 was incubated with [α-³²P]dCTP, ddTTP, and cell extracts. The incubations were performed with wild type (wt) or FEN1 null (FEN1^−/−) DT40 cell extracts. (C) The reaction mixtures with FEN1 null extract were supplemented with 20 and 100 nM purified FEN1 as shown above the gel. (D) The relative amount of LP-BER product (16-mer) was plotted against incubation time.