Cy5 maleimide

Oligonucleotides of Cy3-labeled DNA (Table 1) were obtained from Integrated DNA Technologies (IDT, Coralville, IA). Two complementary strains were mixed and annealed by heating at 80 °C and then slowly cooling to room temperature. The Cy3-labeled DNA was mixed with purified PhoP at a 1:2 DNA-protein molar ratio in the binding buffer (20 mM HEPES, pH 7.5, 100 mM NaCl, and 5 mM MgCl₂). Cy5-maleimide (GE Life Sciences) was dissolved in anhydrous dimethylformamide according to manufacturer’s instructions. Cy5-maleimide in 10-fold excess was mixed with PhoP or PhoP-DNA in the binding buffer supplemented with 1 mM TCEP, and the mixture was incubated at 4 °C overnight. The labeling mixture was first passed through a PD MiniTrap column (GE Healthcare) in the binding buffer to remove the majority of free Cy5 dye; fractions containing the protein were then passed through a Superdex 200 column to further purify the complex. The concentration of the complex was determined by UV-Vis spectrophotometry monitoring absorbance at 260, 280, 550, and 650 nm, which are the absorbance maxima of DNA, protein, Cy3, and Cy5, respectively.

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 9^th 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.