Figure 1.

Inefficient incision at an AP site created after excision of adenine opposite 8-oxoguanine by MUTYH in a cell-free extract. (A) Thymocyte extracts (30 μg protein per lane) prepared from wild-type (lane 1, WT) and MUTYH-null (lane 2, KO) mice were subjected to western blotting with anti-hMUTYH antibody (25). As a positive control, in vitro transcription/translation product (lane 3, mMUTYH) of mouse Mutyh cDNA was applied. (B) Duplex oligonucleotides (20 nM) containing *A:GO (lanes 1–3) or *A:G (lanes 5–7) were incubated with cell-free extracts (90 μg/12.5 μl reaction) prepared from thymocytes of wild-type (lanes 2, 6, WT) and MUTYH-null (lanes 3, 7, KO) mice for 60 min, and reaction products treated with NaOH were fractionated. Lanes 1, 5, no extract (−); lane 4, marker oligonucleotides (M, 19-OH and 19-P). (C) Duplex oligonucleotides containing *A:G (lanes 2–4) *A:GO (lanes 6–8), and *A:T (lanes 10–12) were incubated with (+) or without (−) wild-type cell-free extract as in B, and the reaction mixture treated without (−) or with NaOH (+) was fractionated. The plots obtained by GeneScan are shown. Lanes 1, 5, 9, marker oligonucleotides (19-OH and 19-P). (D) Duplex oligonucleotides containing *A:GO were incubated with wild-type cell-free extracts (60 μg/12.5 μl reaction), for the times noted, and reaction products treated with NaOH were fractionated. Open square, production of 19-P; open triangle, production of 19-OH. Open circle, sum of 19-OH and 19-P. (E) Duplex oligonucleotides containing *A:G were incubated with wild-type cell-free extracts as in (D). Closed square, production of 19-P; closed triangle, production of 19-OH. (F) Preferential action of MUTYH on duplex oligonucleotides containing *A:GO. Results from short-term reactions are shown as in D and E. All data are shown as the means ± S.E.M. of triplicate assays, and the results from one of two independent experiments are presented.