Structural and functional studies on phi 29 DNA polymerase.
The Bacillus subtilis phage phi 29 DNA polymerase, involved in protein-primed viral DNA replication, contains several amino acid consensus sequences common to other eukaryotic-type DNA polymerases. Using site-directed mutagenesis, we have studied the functional significance of a C-terminal conserved region, represented by the Lys-X-Tyr ("K-Y") motif. Single point mutants have been constructed and the corresponding proteins have been overproduced and characterized. Measurements of the activity of the mutant proteins indicated that the invariant Lys and Tyr residues play a critical role in DNA polymerization. Interestingly, substitution of the invariant Lys either by Arg or Thr, produced enzymes with an increased or a largely reduced, respectively, capability to use a protein as primer, an intrinsic property of TP-priming DNA polymerases. On the other hand, the viral protein p6, which stimulates initiation of phi 29 DNA replication by formation of a nucleoprotein complex at both DNA replication origins, increased (about 5-fold) the insertion fidelity of phi 29 DNA polymerase during the formation of the TP-dAMP initiation complex. We propose a model in which the special strategy to maintain the integrity of the phi 29 DNA ends, by means of a "sliding-back" mechanism, could also contribute to increase the fidelity of phi 29 DNA replication.
Blasco
M A
MA
Centro de Biología Molecular (CSIC-UAM), Universidad Autónoma, Madrid, Spain.
Esteban
J A
JA
Méndez
J
J
Blanco
L
L
Salas
M
M
D000595:Amino Acid Sequence; D017103:Bacillus Phages / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D004261:DNA Replication / Q000502:physiology*; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000502:physiology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D014764:Viral Proteins / Q000502:physiology
D014764:Viral Proteins; C040784:terminal protein, Bacillus phage phi29; D004259:DNA-Directed DNA Polymerase