PHI-29 DNA-POLYMERASE RESIDUE LYS383, INVARIANT AT MOTIF-B OF DNA-DEPENDENT POLYMERASES, IS INVOLVED IN DNTP BINDING

Bacteriophage empty set29 DNA polymerase shares with other DNA-depende nt DNA polymerases several regions of amino acid homology along the pr imary structure. Among them, motif B, characterized by the consensus x(3)Kx(6-7)YG (+ being a positively charged amino acid), appears to be specifically conserved in those polymerases that use DNA but not RNA as template. In particular, the lysine residue of this motif is invari ant in all members of DNA-dependent polymerases.In this paper we repor t a mutational analysis of this invariant residue of motif B with the construction and characterization of two mutant proteins in the corres ponding residue (Lys383) of empty set29 DNA polymerase. Mutant protein s (K383R and K383P) were overexpressed, purified and analyzed under st eady-state conditions. In agreement with the modular organization prop osed for empty set29 DNA polymerase, the exonuclease activity was not affected in either mutant protein. Conversely, mutant K383P showed no detectable capacity to incorporate dNTP substrates using either DNA or TP as primer, although its affinity for DNA was not affected. The con servative substitution of Lys383 by arginine (K383R) resulted in a con siderable impairment to use dNTPs, in both processive and non-processi ve DNA synthesis; the K-m for dNTPs being 200-fold higher than that of the wild-type enzyme. Mutant K383R recovered the wild-type polymerase /exonuclease ratio when Mn2+ was used instead of Mg2+ as metal activat or, indicating a distorted binding of the [dNTP-metal] chelate at the mutant enzyme active site. The positive charge at residue Lys383 was a lso critical in the catalysis of deoxynucleotidylation of the terminal protein by empty set29 DNA polymerase. The results obtained suggest a direct role for the lysine residue in motif B in forming an evolution arily conserved DNA templated dNTP binding pocket. Additionally, K383R mutant protein was also affected in the progression from protein-prim ed initiation to DNA elongation, a switch between two modes of priming that characterizes empty set29 DNA replication. (C) 1997 Academic Pre ss Limited.