Abstract
phi 29 DNA polymerase shares with other alpha-like DNA polymerases several 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 polymerases. Mutants in phi 29 DNA polymerase residue Tyr390 of this conserved motif had been previously described to be affected in DNA-dependent dNTP binding. In this paper, the functional significance of this conserved motif is further studied by the analysis of mutants in conserved residues Asn387, Ser388, and Gly391. Residue Phe393 of phi 29 DNA polymerase has also been selected as target for site-directed mutagenesis because of its conservation within the group of alpha-like DNA polymerases from genomes that replicate by a protein-priming mechanism. Mutant N387Y was shown to be affected both in initiation and polymerization reactions, showing 3-fold higher Km value for dATP and more than 11-fold lower Vmax value than the wild-type enzyme in the initiation reaction; moreover, it was affected in enzyme-DNA translocation. Mutant S388G retained initiation and polymerization activities; interestingly, this mutation significantly increased the efficiency of dNTP incorporation in non-templated reactions. Mutation Gly391 to Asp abolished template-primer binding as shown by gel retardation assays; this mutant was drastically affected in template-dependent dNTP incorporation both in initiation and polymerization reactions, but the efficiency of the non-templated phi 29 terminal protein-deoxynucleotidylation was higher than with the wild-type protein. Mutation Phe393 to Tyr severely decreased initial binding to template-primer DNA molecules, resulting in a reduced activity in DNA primer-dependent polymerization reactions but not in phi 29 terminal protein-dependent ones.
Highlights
629 DNA polymerase shares with other a-likeDNA The linear dslDNA of Bacillus subtilis phage 429 replicates polymerases several regions of amino acid similarity. by aprotein-priming mechanism (Salas, 1991) in which a
The conserved region characterized by virally encoded DNA polymerase catalyzes both theformation the amino acid motif ”Kx,NSxYG” has been proposed of the TP .dAMP covalent complex and to form part of the polymerization active siteof a-like its further processive elongation to produce unit-length 429
Intrinsic properties of 429 residue Tyrssoof this conserved motif had been previ- DNA polymerase are its ability to produce strand displaceously described to be affected in DNA-dependentdNTP ment coupled tothe polymerization process and its high binding
Summary
Nucleotides-Unlabeled nucleotides were purchased from Pharmacia P-L Biochemicals. [ C ~ - ~ ~ P ] ~(N40T0 CPiS/mmol), [(u-~’S]~ATP (600 Ci/mmol), and [Y-~’P]AT(P5000 Ci/mmol) were obtained from. The incubation mixture contained, pUC1S; 3.3 x IO6 counts/min/pmol of 3’ end) was used as substrate in 10 pl, 12 mM Tris-HCI, pH 7.5, 1 m~ EDTA, 20 mM ammonium to assay the exonucleolytic activity of wild-type or mutant $29 DNA sulfate, 0.2 ng of hybrid molecules of oligonucleotideSPlc+6 and5’-. Polymerases on Template-PrimerDNA Molecules-Wild-type the 3’ to 5’ exonuclease activity on dsDNA and as primer for non- and mutant 429 DNApolymerases were assayed in filling-in templated dNTP incorporation. Mutant N387Y needed 5-fold more dNTPs concentration than thewild-type polymerase for the first nucleotide addition (Fig. 2; see Table polymerases included in this group(for review, see Blanco et I), indicating a lower efficiency of d N T P incorporation than al., 1991), Ser is variable Single changeswere designed taking into accountsec- Interestingly, mutant F393Y needed even lower dNTPs conondary structure predictions (Chou and Fasman, 1978; Gar- centration than the wild-type DNA polymerase to carry out nier et al, 1978) and the proposals for conservative changes the first polymerization event, probably due to the lower 3’
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