FIGURE 4.

FAN1 cleavage requires ∼10 base pairs of dsDNA on both sides of the flap. A, schematic of the X-12 and X-8 substrates. The tetranucleotide sequence in parentheses is absent from the X-8 substrate. The asterisk indicates the ³²P-labeled phosphate. B, the substrates were incubated with the enzymes at the indicated enzyme:substrate ratio at 37 °C for 1, 10, 30, and 60 min (FAN1-WT, lanes 1–5 and 10–14; FAN1 D960A, lanes 6–9) or 1 and 60 min (lanes 15 and 16). The X-8 substrate was extremely inefficiently processed even at 30 °C (bottom left panel), which indicates that the lack of flap cleavage was not caused by denaturation (denat) of the X structure. (Only the bands indicated by the arrowheads were quantified.) Bottom right panel, 10% native polyacrylamide gel of the two substrates, incubated at 37 °C for 30 min. This additional control experiment shows that both substrates were predominantly annealed under the conditions of the reaction.