Hape1

Oligonucleotides used in this experiment were synthesized from Integrated DNA Technologies (Supplementary Table S1). Forty pmols of 35-nt-long oligonucleotide containing a tetrahydrofuran (THF) at position 18 (F35[AP]) was radiolabeled with [γ-³²P]ATP using T4 Polynucleotide Kinase (Takara) at 37°C for 1.5 h and annealed to the complementary strand to generate the AP site analog substrate. Oligonucleotide substrate (25 nM) was incubated with 5 nM each of MBP-APE1L, -APE2 and -ARP in the AP endonuclease reaction buffer (10 mM Tris-HCl, pH 7.4, 50 mM NaCl, 200 μg/ml BSA, 2.5 mM MgCl₂, 0.5 mM DTT) at 37°C for 30 min. As a reaction control, the same oligonucleotide was reacted with 0.5 unit of hAPE1 (NEB). For kinetics analysis, various amounts of radiolabeled oligonucleotide substrate (0–100 nM) containing a THF was incubated with 5 nM MBP-ARP at 37°C for 4 min. Reaction was terminated by adding 100 mM NaOH and boiling for 10 min. Reactions were denatured and separated on a 15% denaturing polyacrylamide gel. The gel was exposed to a phosphorimager screen (Fujifilm) and the radioactivity was measured using the Fujifilm BAS-5000 phosphorimager.