phi29 DNA polymerase residue Phe128 of the highly conserved (S/T)Lx(2)h motif is required for a stable and functional interaction with the terminal protein.
Bacteriophage phi29 encodes a DNA-dependent DNA polymerase belonging to the eukaryotic-type (family B) subgroup of DNA polymerases that use a protein as primer for initiation of DNA replication. By multiple sequence alignments of DNA polymerases from such a family, we have been able to identify two amino acid residues specifically conserved in the protein-priming subgroup of DNA polymerases, a phenylalanine contained in the (S/T)Lx(2)h motif, and a glutamate belonging to the Exo III motif. Here, we have studied the functional role of these residues in reactions that are specific for DNA polymerases that use a protein-primed DNA replication mechanism, by site-directed mutagenesis in the corresponding amino acid residues, Phe128 and Glu161 of phi29 DNA polymerase. Mutations introduced at residue Phe128 severely impaired the protein-primed replication capacity of the polymerase, being the interaction with the terminal protein (TP) moderately (mutant F128A) or severely (mutant F128Y) diminished. As a consequence, very few initiation products were obtained, and essentially no transition products were detected. Interestingly, phi29 DNA polymerase mutant F128Y showed a decreased binding affinity for short template DNA molecules. These results, together with the high degree of conservation of Phe128 residue among protein-primed DNA polymerases, suggest a functional role for this amino acid residue in making contacts with the TP during the first steps of genome replication and with DNA in the further replication steps.
Rodríguez
Irene
I
Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Universidad Autónoma, Cantoblanco, E-28049 Madrid, Spain.
Lázaro
José M
JM
Salas
Margarita
M
de Vega
Miguel
M
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D017103:Bacillus Phages / Q000201:enzymology* / Q000235:genetics; D017124:Conserved Sequence; D017931:DNA Primers / Q000235:genetics; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010649:Phenylalanine / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D013698:Templates, Genetic; D014764:Viral Proteins / Q000378:metabolism*
D017931:DNA Primers; D014764:Viral Proteins; C040784:terminal protein, Bacillus phage phi29; D010649:Phenylalanine; D004259:DNA-Directed DNA Polymerase