“…To examine the substrate specificity of each glycosylase, 2.5 pmol of a FAM-labelled duplex containing a target pyrimidine pair opposite A or G was incubated with hUNG2 (0.07 mg, MW 40.0 kDa, 1.75 pmol), hTDG (1.5 mg, MW 48.4 kDa, 31 pmol), hSMUG1 (0.5 mg, 30.7 kDa, 16.3 pmol) in buffer containing 10 mM K 2 HPO 4 , 30mM NaCl, 40mM KCl, pH 7.9 or hMBD4 (0.5 mg, 66.9 kDa, 7.5 pmol) in buffer containing 20 mM tris-HCI, 1 mM DTT and 1mM EDTA at pH 8.0 and incubated for 1 h at 37°C. Recombinant hyTDG(Y163K) (0.5 mg, MW 29.7 kDa, 16.8 pmol) with no glycosylase activity, but efficient AP endonuclease activity [ 63 , 64 ], was added and incubated at 65°C for 1 h to cleave the phosphodiester backbone at the abasic site. An equal volume of formamide was added and samples were loaded onto a 20% polyacrylamide gel containing 6M urea and 1xTBE buffer and electrophoresed at 180 V for 40 min using a Bio-Rad Mini-PROTEAN Tetra Cell (Hercules, CA).…”