Phi 29 DNA Polymerase Residue Leu384, Highly Conserved in Motif B of Eukaryotic Type DNA Replicases, Is Involved in Nucleotide Insertion Fidelity

J Biol Chem. 2003 Aug 29;278(35):33482-91. doi: 10.1074/jbc.M303052200. Epub 2003 Jun 12.

Abstract

Replicative DNA polymerases achieve insertion fidelity by geometric selection of a complementary nucleotide followed by induced fit: movement of the fingers subdomain toward the active site to enclose the incoming and templating nucleotides generating a binding pocket for the nascent base pair. Several residues of motif B of DNA polymerases from families A and B, localized in the fingers subdomain, have been described to be involved in template/primer binding and dNTP selection. Here we complete the analysis of this motif, which has the consensus "KLX2NSXYG" in DNA polymerases from family B, characterized by mutational analysis of conserved leucine, Leu384 of phi 29 DNA polymerase. Mutation of Leu384 into Arg resulted in a phi 29 DNA polymerase with reduced nucleotide insertion fidelity during DNA-primed polymerization and protein-primed initiation reactions. However, the mutation did not alter the intrinsic affinity for the different dNTPs, as shown in the template-independent terminal protein-deoxynucleotidylation reaction. We conclude that Leu384 of phi 29 DNA polymerase plays an important role in positioning the templating nucleotide at the polymerization active site and in controlling nucleotide insertion fidelity. This agrees with the localization of the corresponding residue in the closed ternary complexes of family A and family B DNA polymerases, contributing to form the binding pocket for the nascent base pair. As an additional effect, mutant polymerase L384R was strongly reduced in DNA binding, resulting in reduced processivity during polymerization.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Motifs
  • Bacillus Phages / enzymology*
  • Base Sequence
  • Binding Sites
  • Conserved Sequence
  • Crystallography, X-Ray
  • DNA / metabolism
  • DNA Mutational Analysis
  • DNA-Directed DNA Polymerase / chemistry*
  • Electrophoresis, Agar Gel
  • Kinetics
  • Leucine / chemistry*
  • Models, Molecular
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Mutation
  • Protein Binding
  • Protein Structure, Tertiary

Substances

  • DNA
  • DNA replicase
  • DNA-Directed DNA Polymerase
  • Leucine