Phi29 DNA polymerase residues Tyr59, His61 and Phe69 of the highly conserved ExoII motif are essential for interaction with the terminal protein.
Phage Phi29 encodes a DNA-dependent DNA polymerase belonging to the eukaryotic-type (family B) subgroup of DNA polymerases that use a protein as the primer for initiation of DNA synthesis. In one of the most important motifs present in the 3'-->5' exonucleolytic domain of proofreading DNA polymerases, the ExoII motif, Phi29 DNA polymerase contains three amino acid residues, Y59, H61 and F69, which are highly conserved among most proofreading DNA polymerases. These residues have recently been shown to be involved in proper stabilization of the primer terminus at the 3'-->5' exonuclease active site. Here we investigate by means of site-directed mutagenesis the role of these three residues in reactions that are specific for DNA polymerases utilizing a protein-primed DNA replication mechanism. Mutations introduced at residues Y59, H61 and F69 severely affected the protein-primed replication capacity of Phi29 DNA polymerase. For four of the mutants, namely Y59L, H61L, H61R and F69S, interaction with the terminal protein was affected, leading to few initiation and transition products. These findings, together with the specific conservation of Y59, H61 and F69 among DNA polymerases belonging to the protein-primed subgroup, strongly suggest a functional role of these amino acid residues in the DNA polymerase-terminal protein interaction.
Eisenbrandt
Ralf
R
Centro de Biología Molecular Severo Ochoa (C.S.I.C.-U.A.M.), Facultad de Ciencias, 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; D001435:Bacteriophages / Q000201:enzymology*; D020134:Catalytic Domain; D017124:Conserved Sequence; D004247:DNA / Q000378:metabolism; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003838:Deoxyadenine Nucleotides / Q000378:metabolism; D006639:Histidine / Q000235:genetics / Q000502:physiology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000235:genetics / Q000502:physiology; D016415:Sequence Alignment; D014443:Tyrosine / Q000235:genetics / Q000502:physiology; D014764:Viral Proteins / Q000378:metabolism*
D003838:Deoxyadenine Nucleotides; D014764:Viral Proteins; C061994:p3 protein, bacteriophage phi 29; D014443:Tyrosine; D010649:Phenylalanine; D006639:Histidine; D004247:DNA; D004259:DNA-Directed DNA Polymerase