Human ap endonuclease 1 ape1

E. coli uracil DNA glycosylase (UDG, ca# M0280S), human AP endonuclease 1 (APE1, ca# M0282S), and human single-strand selective monofunctional uracil DNA glycosylase (hSMUG1, ca# M0336S) were purchased from New England Biolabs (NEB).

Oligonucleotides used (Supplementary Table S1) were synthesized by Integrated DNA Technologies (IDT) and purified by PAGE or dual HPLC before use. Double-stranded DNA substrates were prepared by mixing a 5 µM solution of a fluorescein (Fl) or alexa (Al) labelled oligonucleotide with a 10 µM solution of an unlabelled complementary oligonucleotide. DNA substrates with one nucleotide gap with a 3′-OH end were prepared by mixing a 5 µM solution of a 5′-alexa (Al) labelled oligonucleotide (Al_28-OH) with a 10 µM solution of both unlabelled oligonucleotide, corresponding to the complementary strand (CGR_G, CGR_A, CGR_T or CGR_C) and to the oligonucleotide containing a 5′-P, 5′-OH or 5′-THF terminus (P_30-51, OH_30-51 or THF_30-51, respectively). Annealing was carried out by heating at 95 °C for 5 min followed by slowly cooling to room temperature. DNA substrates with a 5′-dRP end were generated by incubating an oligonucleotide duplex containing a U:G mispair with 0.5 U of Escherichia coli uracil DNA glycosylase (UDG) (New England Biolabs) and 10 U of human AP endonuclease 1 (APE-1) (New England Biolabs, NEB).