Structure and mechanism of the polynucleotide kinase component of the bacterial Pnkp-Hen1 RNA repair system

Structure-guided mutagenesis. (A) Aliquots (8 μg) of the Ni-Agarose preparations of the indicated His₁₀CthPnkp-(1-425) proteins were analyzed by SDS-PAGE. The polypeptides were visualized by staining with Coomassie blue dye. The positions and sizes (in kilodaltons) of marker polypeptides are indicated. (B) Kinase reaction mixtures contained 180 pmol of 36-mer 5′-OH DNA oligonucleotide and 100 ng of the indicated His₁₀CthPnkp-(1-425) proteins. The extents of label transfer from [γ³²P]ATP to the 36-mer DNA are shown. Each datum in the bar graph is the average of three experiments ±SEM.