PRIMER TERMINUS STABILIZATION AT THE PHI-29 DNA-POLYMERASE ACTIVE-SITE - MUTATIONAL ANALYSIS OF CONSERVED MOTIF KXY

phi 29 DNA polymerase shares with other DNA-dependent DNA polymerases several regions of amino acid homology along the primary structure. A conserved amino acid motif, located in the C-terminal portion of the p olypeptide and characterized by the amino acid sequence KK(K/R)Y, is c onserved in the group of eukaryotic-type DNA polymerases. In the subgr oup of DNA polymerases that have a protein-priming mechanism, this mot if is restricted to the sequence KXY, X never being a positively charg ed amino acid. Residues Lys(498) and Tyr(500) form this conserved moti f in phi 29 DNA polymerase. Mutant K498T, in which the positive charge of the motif has been eliminated, was strongly affected both in initi ation (terminal protein-dAMP formation, using terminal protein as prim er) and DNA polymerization reactions. Mutants K498R and Y500S were abl e to carry out the initiation reaction to a higher or similar extent, respectively, than wild-type phi 29 DNA polymerase but were affected i n DNA polymerization reactions. Ah of the mutations severely affected the stable binding of the polymerase to a primer-template DNA. In addi tion, all of the mutant polymerases analyzed in this work showed an un usually strong 3'-5' exonuclease activity both under polymerization or non-polymerization conditions. The results obtained suggest a role of the conserved residues of the KXY motif in stabilizing the primer ter minus at the polymerization active site, the positive charge of residu e Lys(498) being critical for the synthetic activities of phi 29 DNA p olymerase.