phi29 DNA polymerase-terminal protein interaction. Involvement of residues specifically conserved among protein-primed DNA polymerases.
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.
Rodríguez
Irene
I
Instituto de Biología Molecular "Eladio Viñuela", Centro de Biología Molecular "Severo Ochoa", Universidad Autónoma, Canto Blanco, 28049 Madrid, Spain.
Lázaro
José M
JM
Salas
Margarita
M
De Vega
Miguel
M
D000595:Amino Acid Sequence; D001120:Arginine / Q000378:metabolism; D017103:Bacillus Phages / Q000201:enzymology; D017124:Conserved Sequence; D004247:DNA / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D008239:Lysine / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D014764:Viral Proteins / Q000378:metabolism*
D014764:Viral Proteins; C040784:terminal protein, Bacillus phage phi29; D004247:DNA; D001120:Arginine; D004259:DNA-Directed DNA Polymerase; D008239:Lysine