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Phi 29 DNA polymerase active site. Mutants in conserved residues Tyr254 and Tyr390 are affected in dNTP binding
M A Blasco1, J M Lázaro, A Bernad
1Centro de Biología Molecular, Universidad Autónoma, Canto Blanco, Madrid, Spain.
Abstract:
Phi 29 DNA polymerase shares with other alpha-like DNA polymerases several regions of amino acid similarity. Among them, the two conserved regions characterized by the amino acid motifs "D-NSLYP" and "K--NS(L/V)YG," regions 1 and 2a, respectively, according to Blanco et al. (Blanco, L., Bernad, A., Blasco, M. A. and Salas, M. (1991) Gene (Amst.) 100, 27-38) have been proposed to be part of the polymerization active site of alpha-like DNA polymerases. One phi 29 DNA polymerase mutant in residue Tyr254, located in conserved region 1, and two mutants in residue Tyr390, located in conserved region 2a, have been characterized. The three phi 29 DNA polymerase mutant proteins were affected in polymerization when Mg(2+)-dNTPs were used as substrate. However, when the substrate was Mn(2+)-dNTP, mutants behaved as the wild-type phi 29 DNA polymerase. Mutant Tyr254 to Phe (Y254F) was strongly affected in the protein-primed initiation step of phi 29 DNA replication showing a decreased affinity for Me(2+)-dATP, the initiating nucleotide. Furthermore, the analysis of the template-independent deoxynucleotidylation of the TP by Y254F mutant polymerase is consistent with a change in the relative affinity for dNTPs. On the other hand, mutants Y390F and Y390S were found to be hypersensitive to the dNTP analogs 2-(p-n-butylanilino)dATP and N2-(p-n-butyl-phenyl)dGTP. The results obtained indicate that residues Tyr254 and Tyr390 are involved, directly or indirectly, in Me(2+)-dNTP binding.
Insights
Investigating mutations in phi 29 DNA polymerase reveals that specific tyrosine residues are crucial for magnesium-dependent nucleotide binding and DNA replication. These findings shed light on the enzyme's active site and substrate interactions.
Area of Science:
- Molecular Biology
- Enzymology
- Biochemistry
Background:
- Phi 29 DNA polymerase, an alpha-like DNA polymerase, possesses conserved amino acid regions implicated in its polymerization active site.
- Two key conserved regions, region 1 (motif "D-NSLYP") and region 2a (motif "K--NS(L/V)YG"), are critical for polymerase function.
Purpose of the Study:
- To investigate the role of specific tyrosine residues (Tyr254 and Tyr390) in conserved regions 1 and 2a of phi 29 DNA polymerase.
- To elucidate the involvement of these residues in magnesium-dependent nucleotide binding and DNA replication initiation.
Main Methods:
- Site-directed mutagenesis was used to create phi 29 DNA polymerase mutants: Y254F, Y390F, and Y390S.
- Enzyme activity assays were performed using magnesium-dNTPs and manganese-dNTPs as substrates.
- Analysis of protein-primed initiation and template-independent deoxynucleotidylation was conducted.
Main Results:
- Mutant polymerases showed impaired polymerization with Mg(2+)-dNTPs but normal activity with Mn(2+)-dNTPs.
- The Y254F mutant exhibited reduced affinity for the initiating nucleotide (Mg(2+)-dATP) and altered dNTP affinity during initiation.
- Mutants Y390F and Y390S displayed hypersensitivity to specific dNTP analogs.
Conclusions:
- Residues Tyr254 and Tyr390 are essential for Mg(2+)-dNTP binding in phi 29 DNA polymerase.
- These findings highlight the direct or indirect involvement of these tyrosine residues in substrate recognition and catalytic activity.
- The study provides insights into the mechanism of alpha-like DNA polymerases and their active site structure.