Functional importance of bacteriophage phi29 DNA polymerase residue Tyr148 in primer-terminus stabilisation at the 3'-5' exonuclease active site.
Recent crystallographic resolution of varphi29 DNA polymerase complexes with ssDNA at its 3'-5' exonuclease active site has allowed the identification of residues Pro129 and Tyr148 as putative ssDNA ligands, the latter being conserved in the Kx(2)h motif of proofreading family B DNA polymerases. Single substitution of varphi29 DNA polymerase residue Tyr148 to Ala rendered an enzyme with a reduced capacity to stabilize the binding of the primer terminus at the 3'-5' exonuclease active site, not having a direct role in the catalysis of the reaction. Analysis of the 3'-5' exonuclease on primer/template structures showed a critical role for residue Tyr148 in the proofreading of DNA polymerisation errors. In addition, Tyr148 is not involved in coupling polymerisation to strand displacement in contrast to the catalytic residues responsible for the exonuclease reaction, its role being restricted to stabilisation of the frayed 3' terminus at the exonuclease active site. Altogether, the results lead us to extend the consensus sequence of the above motif of proofreading family B DNA polymerases into Kx(2)hxA. The different solutions adopted by proofreading DNA polymerases to stack the 3' terminus at the exonuclease site are discussed. In addition, the results obtained with mutants at varphi29 DNA polymerase residue Pro129 allow us to rule out a functional role as ssDNA ligand for this residue.
Pérez-Arnaiz
Patricia
P
Instituto de Biología Molecular "Eladio Viñuela", Centro de Biología Molecular "Severo Ochoa", Cantoblanco, Madrid, Spain.
Lázaro
José M
JM
Salas
Margarita
M
de Vega
Miguel
M
D000595:Amino Acid Sequence; D000818:Animals; D017103:Bacillus Phages / Q000201:enzymology / Q000235:genetics; D020134:Catalytic Domain / Q000235:genetics*; D004277:DNA, Single-Stranded; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D004795:Enzyme Stability; D005092:Exonucleases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011392:Proline / Q000378:metabolism; D011485:Protein Binding; D016415:Sequence Alignment; D014443:Tyrosine / Q000378:metabolism*
D004277:DNA, Single-Stranded; D014443:Tyrosine; D011392:Proline; D004259:DNA-Directed DNA Polymerase; D005092:Exonucleases