Research / 2019 / Article / Fig 2

Research Article

Mechanism of DNA Lesion Homing and Recognition by the Uvr Nucleotide Excision Repair System

Figure 2

Entrapment strategy to form a preincision complex. (a) Schematic describing the disulfide crosslinking (DXL) and stepwise assembly strategy. (b) Details of the trapping chemistry. A cysteine residue engineered into UvrB attacks a disulfide-bearing tether attached to the N4-position of a cytosine. Curved arrows denote electron flow in the crosslinking reaction. (c) UvrC incision assays on the crosslinked UvrB-dsDNA complexes were analyzed on 15% denaturing polyacrylamide gel. 5′ ends of both inner and outer strands are radioactively labeled. Lanes 1-4 were cropped from the same experiment. Lanes 5 and 6 (with DNA ladder) were run on 23.5% denaturing polyacrylamide gels for better resolution. (d) DNA sequence used for this study and experimentally determined 5′ and inferred 3′ incision sites. The lesion-mimetic cytosine, located 8-nucleotide downstream on the inner strand, is shown in red.