PHI-29 DNA-POLYMERASE ACTIVE-SITE - RESIDUE ASP(249) OF CONSERVED AMINO-ACID MOTIF DX(2)SLYP IS CRITICAL FOR SYNTHETIC ACTIVITIES

Phi29 DNA polymerase shares with other alpha-like DNA polymerases seve ral regions of amino acid sequence similarity and sensitivity to inhib itors of eukaryotic DNA polymerase alpha. In this paper, site-directed mutants in the phi29 DNA polymerase residues Asp249, Ser252, Leu253, and Pro255 of the conserved amino acid motif ''Dx2SLYP'' are described . Two mutants, D249E and S252R, were drastically affected in all the s ynthetic activities, whereas their 3' to 5' exonuclease activity and i nteraction with the TP primer was normal. Mutant D249E, slightly affec ted in template-primer binding, was completely inactive in all conditi ons tested, suggesting that Asp249 Could be playing a direct role in c atalysis. On the other hand, mutant S252R, strongly affected in templa te-primer binding, showed some DNA polymerization activity in the pres ence of Mn2+. Mutants S252G and P255S showed a reduced template-primer binding ability; these mutants, together with mutant L253V, showed me tal ion-dependent phenotypes in their synthetic activities and altered sensitivities to the PP(i) analog phosphonoacetic acid. All these res ults support the hypothesis that the Dx2SLYP motif forms part of the p olymerization active site of the phi29 DNA polymerase, being the Asp24 9 residue critical both for protein-primed initiation and DNA polymeri zation.