We selected a catalytically active mutant of Dpo4 used in previous work with mutations in the Finger domain (C31S, N70C) for the present study.(25 (link)–27 (link)) The engineered Cys mutation allowed for site-specific fluorophore labeling (Figure 1B ) which was carried out by incubation of Dpo4 with a 15-fold molar excess of Cy5-maleimide (GE Healthcare), overnight at 4°C in a buffer containing 50 mM Tris (pH 7.2), 150 mM NaCl, 0.5 mM TCEP, and 10% glycerol. Unincorporated free dye was then removed with Micro Bio-Spin columns (Bio-Rad). By measuring the absorbance at 280 nm and 650 nm, the ratio of protein concentration to dye concentration revealed a labelling efficiency of 91% for the reaction.
The 21-mer primer containing a 5′-biotin for surface immobilization as well as a 5-C6-amino-2′-deoxythymidine modification at the 9th base from the 3′terminus for Cy3-NHS-ester labelling, and control undamaged template were purchased from Integrated DNA Technologies. This labelling was performed according to the manufacturer’s protocol (GE Healthcare) (Figure 1A ). The oligonucleotide containing the 8-oxo-dG base was purchased from Midland Certified Reagent Company, Incorporated. The primer and template oligonucleotides were annealed to each other by heating to 80°C for 5 minutes followed by slow cooling to room temperature.
The 21-mer primer containing a 5′-biotin for surface immobilization as well as a 5-C6-amino-2′-deoxythymidine modification at the 9th base from the 3′terminus for Cy3-NHS-ester labelling, and control undamaged template were purchased from Integrated DNA Technologies. This labelling was performed according to the manufacturer’s protocol (GE Healthcare) (
