Abstract
phi 29 DNA polymerase shares with other alpha-like DNA polymerases several regions of amino acid sequence similarity and sensitivity to inhibitors of eukaryotic DNA polymerase alpha. In this paper, site-directed mutants in the phi 29 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 synthetic activities, whereas their 3' to 5' exonuclease activity and interaction with the TP primer was normal. Mutant D249E, slightly affected in template-primer binding, was completely inactive in all conditions tested, suggesting that Asp249 could be playing a direct role in catalysis. On the other hand, mutant S252R, strongly affected in template-primer binding, showed some DNA polymerization activity in the presence of Mn2+. Mutants S252G and P255S showed a reduced template-primer binding ability; these mutants, together with mutant L253V, showed metal ion-dependent phenotypes in their synthetic activities and altered sensitivities to the PPi analog phosphonoacetic acid. All these results support the hypothesis that the Dx2SLYP motif forms part of the polymerization active site of the phi 29 DNA polymerase, being the Asp249 residue critical both for protein-primed initiation and DNA polymerization.
Highlights
From the Centro deBiologia Molecular “SeueroOchoa” (Consejo Superior de Inuestigaciones Cientificas- Uniuersidad Autonoma de Madrid), Universidad Autonoma, Cantoblanco, 28049 Madrid, Spain
Mutant D249E, slightlyaffected in template-primerbinding, was completely inactive in all conditions tested, 429 DNA polymerase has been included in thegroup of alike DNA polymerases because of its sensitivity to specific inhibitors of eukaryotic DNApolymerase a (Blanco and Salas, 1986; Bernad et al, 1987) and because it contains several regions of amino acid similarity conserved among thesegroup of polymerases (Larder et al, 1987a; Bernad et al, 1987;Wong et al, 1988; Blanco et al, 1991; ItoandBraithwaite, 1991; suggesting that Aspz4’ could beplaying adirect role in Braithwaite and Ito, 1993) that have been proposed to form catalysis
Mutant S252R, strongly the polymerization active site (Larder et al, 1987a; Gibbs et affected in template-primer binding, showed some al., 1988).This hypothesisis supported by the fact thasteveral
Summary
From the Centro deBiologia Molecular “SeueroOchoa” (Consejo Superior de Inuestigaciones Cientificas- Uniuersidad Autonoma de Madrid), Universidad Autonoma, Cantoblanco, 28049 Madrid, Spain. Reaction Pro255form conserved motif Dx2SLYP in 429 DNA polymermixtures contained, in 10pl, 12 mM Tris-HC1 (pH 7.5), 1 mM EDTA, 20 mM ammonium sulfate, 0.2 ng of hybrid molecules of oligonucleotide SPlc+6 (21-mer), and 5’-32P-labeledoligonucleotide SP1 (15mer) and the indicated amount of either wild-type or mutant $29 DNA polymerase.
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