Figure 4.

Nuclease and ICL-unhooking activity of the WT and the mutants of PaFAN1. A, time-dependent nuclease activity of WT (50 nm, lanes 2–5) and mutant PaFAN1 (50 nm, lanes 6–9; R228A, lanes 10–13; K260A, lanes 14–17; R228A/K260A) toward a 1-nt 5′ flap DNA (10 nm). DNA was labeled at 3′ end of a 5′ flap strand (red asterisk on top). Initial cleavage products are shown at 12- and 13-nt positions, which are further cleaved by exonuclease activity. B, quantified fraction product in each reaction. Data are represented as means ± S.D. C, time-dependent ICL-unhooking activity of WT PaFAN1 (250 nm, lanes 2–4) and mutants (250 nm, lanes 5–7; R228A, lanes 8–10; K260A, lanes 11–13; R228A/K260A) toward ICL-3/G3 (25 nm, top). Fluorescence signals were scanned using two wavelengths of 488 and 532 nm for FAM (left panel) and HEX or CY3 (right panel), respectively. D, time-dependent ICL-unhooking activity of various PaFAN1 proteins toward ICL-9/G3. E, graph showing the quantified fraction product “b” (left) or “e” (right) in C and F in reaction in D.