phi 29 DNA Polymerase Active Site: Role of Residue Val250 as Metal-dNTP Complex Ligand and in Protein-Primed Initiation

作者：Perez Arnaiz Patricia; Lazaro Jose M; Salas Margarita^*; de Vega Miguel

来源：Journal of Molecular Biology, 2010, 395(2): 223-233.

DOI：10.1016/j.jmb.2009.10.061

• phi 29 DNA polymerase
• metal ligand
• polymerisation active site
• protein-priming
• strand displacement

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 phi 29 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 phi 29 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 phi 29 DNA replication because of a large reduction in the catalytic efficiency of the protein-primed reactions. In the light of the phi 29 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.

• 出版日期2010-1-15