PHI-29 DNA-POLYMERASE ACTIVE-SITE - THE CONSERVED AMINO-ACID MOTIF KX3NSXYG IS INVOLVED IN TEMPLATE-PRIMER BINDING AND DNTP SELECTION

Phi29 DNA polymerase shares with other alpha-like DNA polymerases seve ral regions of amino acid similarity. Among them, the conserved region characterized by the amino acid motif ''Kx3NSxYG'' has been proposed to form part of the polymerization active site of alpha-like DNA polym erases. Mutants in phi29 DNA polymerase residue Tyr390 of this conserv ed motif had been previously described to be affected in DNA-dependent dNTP binding. In this paper, the functional significance of this cons erved motif is further studied by the analysis of mutants in conserved residues Asn387, Ser388, and Gly391. Residue Phe393 of phi29 DNA poly merase has also been selected as target for site-directed mutagenesis because of its conservation within the group of alpha-like DNA polymer ases from genomes that replicate by a protein-priming mechanism. Mutan t N387Y was shown to be affected both in initiation and polymerization reactions, showing 3-fold higher K(m) value for dATP and more than 11 -fold lower V(max) value than the wild-type enzyme in the initiation r eaction; moreover, it was affected in enzyme-DNA translocation. Mutant S388G retained initiation and polymerization activities; interestingl y, this mutation significantly increased the efficiency of dNTP incorp oration in non-templated reactions. Mutation Gly391 to Asp abolished t emplate-primer binding as shown by gel retardation assays; this mutant was drastically affected in template-dependent dNTP incorporation bot h in initiation and polymerization reactions, but the efficiency of th e non-templated phi29 terminal protein-deoxynucleotidylation was highe r than with the wild-type protein. Mutation Phe393 to Tyr severely dec reased initial binding to template-primer DNA molecules, resulting in a reduced activity in DNA primer-dependent polymerization reactions bu t not in phi29 terminal protein-dependent ones.