Domain structure and mutational analysis of T4 polynucleotide kinase

T4 polynucleotide kinase (Pnk) is the founding member of a family of 5'-kinase/3'-phosphatase enzymes that heal broken termini in RNA or DNA by converting 3'-PO4/5'-OH ends into 3'-OH/5'-PO4 ends, which are then suitable for sealing by RNA or DNA ligases. Here we employed site directed mutagenesis and biochemical methods to dissect the domain structure of the homotetrameric T4 Pnk protein and to localize essential constituents of the apparently separate active sites for the 5'-kinase and 3'-phosphatase activities. We characterized deletion mutants Pnk(42-301) and Pnk(1-181) which correspond to domains defined by proteolysis with chymotrypsin. Pnk(1-181) is a monomer with no 3'-phosphatase and low residual 5'-kinase activity, Pnk(42-301) is a dimer with no 5'-kinase and low residual 3'-phosphatase activity, Four classes of missense mutational effects were observed, (i) Mutations K15A, S16A, and D35A inactivated the 5'-kinase but did not affect the 3'-phosphatase or the tetrameric quaternary structure of T4 Pnk. 5'-kinase activity was ablated by the conservative mutations K15R, K15Q, and D35N; however, kinase activity was restored by the S16T change. (ii) Mutation D167A inactivated the 3'-phosphatase without affecting the 5'-kinase or tetramerization. (iii) Mutation D85A caused a severe decrement in 5'-kinase activity and only a modest effect on the 3'-phosphatase; the nearby N87A mutation resulted in a significantly reduced 3'-phosphatase activity and slightly reduced 5'-kinase activity. D85A and N87A both affected the quaternary structure, resulting in a mixed population of tetramer and dimer species, (iv) Alanine mutations at 11 other conserved positions had no significant effect on either 5'-kinase or 3'-phosphatase activity.