Acidic Residues and a Predicted, Highly Conserved α-Helix Are Critical for the Endonuclease/Strand Separation Functions of Bacteriophage λ's TerL.

Complementation, endonuclease, strand separation, and packaging assays using mutant TerL(lambda)'s, coupled with bioinformatic information and modeling of its endonuclease, identified five residues, D401, E408, D465, E563, and E586, as critical acidic residues of TerL(lambda)'s endonuclease. Studies of phage and viral TerL nucleases indicate acidic residues participate in metal ion-binding, part of a two-ion metal catalysis mechanism, where metal ion A activates a water for DNA backbone hydrolysis. Modeling places D401, D465, and E586 in locations analogous to those of the metal-binding residues of many phage and viral TerLs. Our work leads to a model of TerL(lambda)'s endonuclease domain where at least three acidic residues from a similar to 185 residue segment (D401 to E586) are near each other in the structure, forming the endonuclease catalytic center at cosN, the nicking site. DNA interactions required to bring the rotationally symmetric cosN precisely to the catalytic center are proposed to rely on an similar to 60 residue region that includes a conserved alpha-helix for dimerization. Metal ion A, positioned by TerL(lambda)'s acidic D401 and E586, would be placed at cosN for water activation, ensuring high accuracy for DNA backbone hydrolysis.