Supplementary Figure 4: Arrangement of protein and DNA molecules in the 3′ overhang–T5Fen D155K complex.

(a) Arrangement of three adjacent asymmetric units. Each consists of one protein and one identical DNA (3ov6) molecule (PDB code 5HP4). Two molecules of oligonucleotide (3ov6₁, orange; 3ov6₂, blue) form a partially base-paired duplex contacting the central T5Fen molecule (grey surface) while the 3´ end of a third (3ov6₃, magenta) threads through its helical arch (helices h4 and h5). (b) The central T5Fen protein from (a) showing nucleotides from two adjacent AUs that contact it. The strands have been labeled X, Y and Z. Strand X passed through the helical arch. The resulting assembly resembles a pseudo-product complex such as could have been derived from hydrolysis of the branched substrate at the indicated phosphodiester (inset, red arrow) – or conceptually, by joining strands X and Z to form the cyan strand. (c) The sequence of partially complementary oligonucleotide 3ov6 shown as a duplex. (d) Comparison of position of divalent metal ions in T5Fen (grey cartoon) and the D155K variant (green cartoon). Grey stick residues indicate ligands for Mg²⁺ ions in M1 and M2 (grey spheres 1 and 2) in the wt protein. In T5FenD155K DNA–Ca²⁺ complex calcium is positioned at M1 and the ɛ-amino group of Lys155 (blue sphere) is situated close to the M2 site making electrostatic interactions with Asp153 and Asp130. (e) The H3TH motif (a.a. 191–225, grey cartoon) of T5Fen binds a potassium ion (magenta sphere) which in turn binds the phosphate group of dT5 in the duplex DNA. Sequence alignment of the H3TH motifs: residues 191-224 of T5FEN; 163-197 ExoIX and 219-252 from hFEN1 shown below. Consensus sequence shown with similar (:), hydrophilic (%) and not dissimilar (.) residues indicated. (f) Interactions between the 3´ end of one DNA strand with helix 1 (h1). Hydrogen bonds indicated by yellow dashes with water molecules as red spheres. Grey sticks indicate amino acids interacting with DNA.