FEN1 flap cleavage on the substrates containing a 5′-phosphate or 5′-THF residue with a 1-nt gap opposite a template 5′R-cdA or 5′S-cdA was examined as described in Materials and Methods. Lanes 1, 4, 7 and 10 represent substrates only. Lanes 2, 5, 8 and 11 correspond to reaction mixtures with 10 nM FEN1. Lanes 3, 6, 9 and 12 correspond to reaction mixtures with 10 nM FEN1 in the presence of 5 nM pol β. Substrates were 32P-labeled at the 3′-end of the downstream primer as indicated. Substrates are illustrated schematically above the gel.