Fig. 6.
EN1 and Exo1 use multiple mechanisms for substrate specificity. A) FEN1 and Exo1 use a hydrophobic wedge to block the path of the duplex DNA. B) FEN1 and Exo1 bind primarily to the complementary strand in two sections, the active site and the K+/H2/3TH. These regions are separated by 24 Å or a helical turn apart. C) The active sites, with seven invariantly conserved carboxylates and two catalytic metals, are protected by a helical gateway and a helical cap. The helical gateway could select for ssDNA or ssRNA. The terminal nucleotide stacks against an aromatic residue and is contacted by two conserved basic residues. D) A double base unpairing in the mechanisms is suggested by the observation of +1 and −1 paired nucleotides in the substrate and a −1 unpaired nucleotide in the product. The scissile phosphate is distant (5 Å) from the catalytic metals when the nucleotides are paired in the substrate structure, but is within catalytic distance (2.2 Å) in the product structure.