phi29 DNA polymerase-terminal protein interaction. Involvement of residues specifically conserved among protein-primed DNA polymerases

J Mol Biol. 2004 Apr 2;337(4):829-41. doi: 10.1016/j.jmb.2004.02.018.


By multiple sequence alignments of DNA polymerases from the eukaryotic-type (family B) subgroup of protein-primed DNA polymerases we have identified five positively charged amino acids, specifically conserved, located N-terminally to the (S/T)Lx(2)h motif. Here, we have studied, by site-directed mutagenesis, the functional role of phi29 DNA polymerase residues Arg96, Lys110, Lys112, Arg113 and Lys114 in specific reactions dependent on a protein-priming event. Mutations introduced at residues Arg96, Arg113 and Lys114 and to a lower extent Lys110 and Lys112, showed a defective protein-primed initiation step. Analysis of the interaction with double-stranded DNA and terminal protein (TP) displayed by mutant derivatives R96A, K110A, K112A, R113A and K114A allows us to conclude that phi29 DNA polymerase residue Arg96 is an important DNA/TP-ligand residue, essential to form stable DNA polymerase/DNA(TP) complexes, while residues Lys110, Lys112 and Arg113 could be playing a role in establishing contacts with the TP-DNA template during the first step of DNA replication. The importance of residue Lys114 to make a functionally active DNA polymerase/TP complex is also discussed. These results, together with the high degree of conservation of those residues among protein-primed DNA polymerases, strongly suggest a functional role of those amino acids in establishing the appropriate interactions with DNA polymerase substrates, DNA and TP, to successfully accomplish the first steps of TP-DNA replication.

Publication types

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

MeSH terms

  • Amino Acid Sequence
  • Arginine / metabolism
  • Bacillus Phages / enzymology
  • Conserved Sequence
  • DNA / metabolism
  • DNA-Directed DNA Polymerase / genetics
  • DNA-Directed DNA Polymerase / metabolism*
  • Lysine / metabolism
  • Molecular Sequence Data
  • Mutation
  • Viral Proteins / metabolism*


  • Viral Proteins
  • terminal protein, Bacillus phage phi29
  • DNA
  • Arginine
  • DNA-Directed DNA Polymerase
  • Lysine