Figure 6.

FEN1 cleavage of CAG repeat double-flap intermediates. FEN1 cleavage of CAG repeat double-flap intermediates resulting from APE1 incision at a CAG hairpin loop region was examined with a substrate containing a 3′-(CAG)₄ and a 5′-THF-(CAG)₃ (left panel) and a substrate with a 3′-(CAG)₇ flap and a 5′-THF-(CAG)₇ flap (right panel). Lanes 1 and 8 correspond to substrates only. Lanes 2 and 9 correspond to reaction mixtures with 250 nM Mus81/Eme1. Lanes 3–4 and 10–11 correspond to reaction mixtures with or without 250 nM Mus81/Eme1 in the presence of 10 nM FEN1. Lanes 5–6 and 12–13 correspond to reaction mixtures with and without 250 nM Mus81/Eme1 in the presence of 10 nM FEN1 and 5 nM pol β. Lanes 7 and 14 correspond to synthesized size markers (M). Substrates were ³²P-labeled at the 3′-end of their downstream strands. A scheme that indicates FEN1 cleavage activity in removing a double-flap with a 5′-THF is illustrated below the gels.