Endonuclease 3

250 ng of each construct were digested with Formamidopyrimidine DNA glycosylase (Fpg, New England Biolabs, Cat. #M0240S) using 1 μL of Fpg (8 units) in the presence of BSA, according to the manufacturer’s instructions, in 1X NEBuffer 1 for 1 hr at 37°C. Products were separated and visualized on 0.7% agarose TBE gels containing ethidium bromide. For the Endonuclease III (Nth) assay, Endonuclease III (New England Biolabs, Cat. # M0268S) in 1X Endonuclease III reaction buffer was used instead.

Bifunctional DNA glycosylases have base removal glycosylase activity and AP lyase activity that leading to a break in the DNA backbone leaving a 5’-phosphate and either a 3’-UA, via α-elimination, or a 5’-phosphate via β,δ-elimination (Fig. 2A). To confirm the presence of glycosylase and AP lyase activity in AGOG, a 20 μ L reaction containing 20 nM 8oxoG:C dsDNA and 100 nM AGOG in 1x Thermopol Buffer was incubated at 65 °C for 30 min. For a β,δ-elimination positive control, the above reaction was performed with Fpg instead of AGOG. For a β-elimination positive control, 20 nM dU:G dsDNA and 100 nM of Uracil DNA glycosylase (New England Biolabs, Ipswich, MA) and Endonuclease III (New England Biolabs, Ipswich, MA) in IX Thermopol Buffer were incubated at 37 °C for 30 min [8 (link),46 (link)]. All reactions were quenched by the addition of equal volume of 85 % formamide and 50 mM EDTA, followed by dilution in water to bring the final concentration of DNA to 2 nM. A 3730x1 Genetic Analyzer (Applied Biosystems) was used for capillary electrophoresis and the resultant fluorescent peaks were analyzed using Peak Scanner software version 1.0 (Applied Biosystems) [42 (link)].