phi29 DNA polymerase active site: role of residue Val250 as metal-dNTP complex ligand and in protein-primed initiation.
DNA polymerases require two acidic residues to coordinate metal ions A and B at their polymerisation active site during catalysis of nucleotide incorporation. Crystallographic resolution of varphi29 DNA polymerase ternary complex showed that metal B coordination also depends on the carbonyl group of Val250 that belongs to the highly conserved Dx(2)SLYP motif of eukaryotic-type (family B) DNA polymerases. In addition, multiple sequence alignments have shown the specific conservation of this residue among the DNA polymerases that use a protein as primer. Thus, to ascertain its role in polymerisation, we have analysed the behaviour of single mutations introduced at the corresponding Val250 of varphi29 DNA polymerase. The differences in nucleotide binding affinity shown by mutants V250A and V250F with respect to the wild-type DNA polymerase agree to a role for Val250 as a metal B-dNTP complex ligand. In addition, mutant V250F was severely affected in varphi29 DNA replication because of a large reduction in the catalytic efficiency of the protein-primed reactions. In the light of the varphi29 DNA polymerase structures, a role for Val250 residue in the maintenance of the proper architecture of the enzyme to perform the protein-primed reactions is also proposed.
Pérez-Arnaiz
Patricia
P
Instituto de Biología Molecular Eladio Viñuela (CSIC), Centro de Biología Molecular Severo Ochoa (CSIC-UAM), C/Nicolás Cabrera 1, Cantoblanco, 28049 Madrid, Spain.
Lázaro
José M
JM
Salas
Margarita
M
de Vega
Miguel
M
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D017103:Bacillus Phages / Q000201:enzymology* / Q000235:genetics; D020134:Catalytic Domain / Q000235:genetics; D004261:DNA Replication; D004279:DNA, Viral / Q000096:biosynthesis / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003854:Deoxyribonucleotides / Q000378:metabolism; D008024:Ligands; D008670:Metals / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D004279:DNA, Viral; D003854:Deoxyribonucleotides; D008024:Ligands; D008670:Metals; D011994:Recombinant Proteins; D004259:DNA-Directed DNA Polymerase