5 alexa fluor 488 fluorescent dye labelled ssdna substrate of 40 bases

The deamination assay described by Byeon et al.27 (link) was used. Whole-cell lysates were prepared using M-PER reagent (Thermo Fisher) with 1 × Halt protease and phosphatase inhibitor cocktail (Thermo Fisher). Briefly, 180 nM 5′ Alexa Fluor 488 fluorescent dye-labelled ssDNA substrate of 40 bases (Integrated DNA Technologies) was incubated at 37 °C for an hour with 10 μl lysate and 10 units of E. coli uracil DNA glycosylase (New England Biolabs) in 10 mM Tris (pH 8.0), 50 mM NaCl, 1 mM dithiothreitol (DTT) and 1 mM EDTA in a volume of 50 μl. The reaction was stopped by adding 40 μg proteinase K (Life Technologies) and incubating it for 20 min at 65 °C. Ten microlitres of 1 N NaOH was added to the reaction, which was then incubated at 37 °C for 15 min. After adding 10 μl of 1 N HCl, the reaction (10 μl) was electrophoresed on a 10% denaturing polyacrylamide gel. Typhoon 9400 Imager (GE Healthcare) was used to scan the gel in fluorescence mode.

2 μM of 5′ Alexa Fluor 488 fluorescent dye-labelled ssDNA substrate of 40 bases (5′-ATTATTATTATTATTATTATTCCCAGGATTTATTTATTTA-3′) (Integrated DNA Technologies) was incubated at 37 °C for an hour with 100 nM or 1 μM r A3G (Origene) that was either pre-treated or untreated with Ribonuclease A (Sigma Aldrich) and 2 units of E. coli uracil DNA glycosylase (New England Biolabs) in 10 mM Tris (pH 8.0), 50 mM NaCl, 1 mM DTT and 1 mM EDTA in a volume of 10 μl.1 μl of 1 N NaOH was added to the reaction, which was then incubated at 37 °C for 15 min. After adding 1 μl of 1N HCl and 12 μl of 2X loading buffer (80% formamide, 10X TBE), the samples were incubated at 65 °C for 5 min. 10 μl of the reaction was electrophoresed on a 10% Mini-PROTEAN TBE-Urea gels (Bio-Rad). Typhoon 9400 Imager (GE Healthcare) was used to scan the gel in fluorescence mode at 488 nm.