phi29 DNA polymerase residue Ser122, a single-stranded DNA ligand for 3'-5' exonucleolysis, is required to interact with the terminal protein.
Three amino acid residues highly conserved in most proofreading DNA polymerases, a phenylalanine contained in the Exo II motif and a serine and a leucine belonging to the S/TLx2h motif, were recently shown to be critical for 3'-5' exonucleolysis by acting as single-stranded DNA ligands (de Vega, M., Lázaro, J.M., Salas, M. and Blanco, L. (1998) J. Mol. Biol. 279, 807-822). In this paper, site-directed mutants at these three residues were used to analyze their functional importance for the synthetic activities of phi29 DNA polymerase, an enzyme able to start linear phi29 DNA replication using a terminal protein (TP) as primer. Mutations introduced at Phe65, Ser122, and Leu123 residues of phi29 DNA polymerase severely affected the replication capacity of the enzyme. Three mutants, F65S, S122T, and S122N, were strongly affected in their capacity to interact with a DNA primer/template structure, suggesting a dual role during both polymerization and proofreading. Interestingly, mutant S122N was not able to maintain a stable interaction with the TP primer, thus impeding the firsts steps (initiation and transition) of phi29 DNA replication. The involvement of Ser122 in the consecutive binding of TP and DNA is compatible with the finding that the TP/DNA polymerase heterodimer was not able to use a DNA primer/template structure. Assuming a structural conservation among the eukaryotic-type DNA polymerases, a model for the interactions of phi29 DNA polymerase with both TP and DNA primers is presented.
de Vega
M
M
Centro de Biología Molecular "Severo Ochoa", Universidad Autónoma, Canto Blanco, 28049 Madrid, Spain.
Blanco
L
L
Salas
M
M
D000595:Amino Acid Sequence; D017103:Bacillus Phages / Q000201:enzymology*; D001483:Base Sequence; D017931:DNA Primers; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003838:Deoxyadenine Nucleotides / Q000378:metabolism; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000378:metabolism; D006868:Hydrolysis; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000378:metabolism*; D014764:Viral Proteins / Q000378:metabolism*
D017931:DNA Primers; D004277:DNA, Single-Stranded; D003838:Deoxyadenine Nucleotides; D008024:Ligands; D014764:Viral Proteins; C040784:terminal protein, Bacillus phage phi29; D012694:Serine; C012873:2'-deoxy-5'-adenosine monophosphate; D004259:DNA-Directed DNA Polymerase; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V