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Bacteriophage Phi 29 DNA Polymerase: An Outstanding Replicase

AutorVega, Miguel de ; Salas, Margarita
Palabras claveDNA Polymerase
Bacillus Subtilis
Phage phi29
Fecha de publicación2009
EditorNova Science Publishers
CitaciónBacterial DNA, DNA Polymerase and DNA Helicases: 329-351 (2009)
ResumenDue to the limited processivity of replicative DNA polymerases (replicases), as well as to their incapacity to unwind parental duplex DNA to allow replication fork progression, their replication efficiency depends on the functional assistance of accessory proteins as processivity factors and helicases. In addition, the inability of DNA polymerases to start de novo DNA synthesis requires the use of a short RNA/DNA molecule to provide the 3’-OH group required to initiate DNA replication. This requisite for a primer creates a dilemma to replicate the ends of linear genomes: once the last primer for the lagging strand synthesis is removed, a portion of ssDNA at the end of the genome will remain uncopied. Bacteriophage φ29 has overcome these issues by means of the unique catalytic features of an outstanding enzyme, the φ29 DNA polymerase. This replicase belongs to the family B (eukaryotic-type) of DNA-dependent DNA polymerases and has served as model to understand the enzymology of these polymerases. As most of the family B members, φ29 DNA polymerase contains both 3´- 5´ exonuclease and polymerization activities residing in two structurally independent domains. During two decades, site-directed mutagenesis studies of individual residues contained in regions of high amino acid similarity have provided the functional insights of this enzyme, extrapolative to other family B members. However, φ29 DNA polymerase is endowed with two distinctive features: high processivity and strand displacement capacity that allow it to replicate the viral genome from a single binding event, without requiring the assistance of unwinding and processivity factors. Recent crystallographic resolution of the structure of the apo and binary/ternary complexes of φ29 DNA polymerase, together with the biochemical studies of site-directed mutants, have given insights into the structural basis responsible for the coordination of the processive polymerization and strand displacement. In addition, such structures have provided the mechanism of translocation of family B DNA polymerases. Another difference with respect to the rest of replicases is the ability of φ29 DNA polymerase to use a protein (terminal protein, TP) as primer, circumventing the end replication problem. Recent resolution of the structure of the φ29 DNA polymerase/TP heterodimer, together with the biochemical analysis of chimerical DNA polymerases and TPs, have given the clues of the specificity of the interaction between both proteins, suggesting a model for the transition from initiation to elongation. We will also discuss how the basic research on the φ29 DNA polymerase properties have led to the development of DNA amplification technologies based on this outstanding enzyme.
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