FIGURE 5.

MUTYH binding and MUTYH/RNase H2 competition. A, sequence of the DNA duplexes used for band-shift experiments and incision assays. B, band-shift experiments were performed on 8-oxodG:dA/rA substrates (30 nm) and increasing MUTYH concentration (1–10 nm) for 1 h at 4 °C in a 10-μl reaction volume. Products were electrophoresed on 8% nondenaturing polyacrylamide gels. C, RNase H2 incision assay. DNA duplex substrate (10 nm) (lane 1) was reacted with RNase H2 enzyme (1 unit) for 1 h at 37 °C without (lane 2) or with a previous incubation with MUTYH (10 nm) for 10 (lane 3) and 30 (lane 4) min. Products were separated by denaturing PAGE and data analysis was performed as described before.