Figure 4.

Photoaffinity labeling of BER proteins in MEF extract by a binary system of photoaffinity reagents, DNA₂ and Pyr-dUTP. (A) A scheme illustrating synthesis of the BER intermediate DNA₂ in cellular extract, as described in the Materials and Methods. (B) The reaction mixtures were incubated with DNA₁ and then irradiated with 365 nm UV light. Absence of FAB-dCTP and Pyr-dUTP (lane 1); presence of FAB-dCTP (lane 2); presence of FAB-dCTP and Pyr-dUTP (lane 3). The reaction mixture was incubated with DNA₁ and irradiated with 312 nm UV light: FAB-dCTP without Pyr-dUTP (lane 4). BER reaction was reconstituted with DNA₁ and purified BER proteins, FEN-1, β-pol and APE (lanes 5–8). After incubation for 30 min at 25°C, the reaction mixture was irradiated with 365 nm UV light (lanes 5 and 6) or 312 nm UV light (lanes 7 and 8). Reaction was performed without Pyr-dUTP (lanes 5 and 7) or with Pyr-dUTP (lanes 6 and 8). The reaction products were separated by 10% SDS–PAGE followed by autoradiography. The positions of protein markers and crosslinked proteins are indicated on the left and right margins, respectively.