Bacteriophage Graphic29 DNA polymerase, the product of the viral gene 2, was originally characterized as a protein involved in the initiation of Graphic29 DNA replication based on both in vivo(1) and in vitro(2, 3, 4) studies. The cloning of gene 2(5), the overproduction and purification of its product(6), and the development of an in vitro system for complete Graphic29 DNA replication (7) allowed the characterization of protein p2 as the viral DNA replicase(8). This monomeric enzyme, with a molecular mass of only about 66 kDa, catalyzes two distinguishable synthetic reactions: 1) DNA polymerization, as any other DNA-dependent DNA polymerase, with insertion discrimination values ranging from 104 to 106 and with an efficiency of mismatch elongation 105-106-fold lower than that of a properly paired primer terminus(9); 2) terminal protein (TP) 1deoxynucleotidylation, which consists of the formation of a covalent linkage (phosphoester) between the hydroxyl group of a specific serine residue (SerGraphic) in Graphic29 TP and 5′-dAMP, requires the presence of divalent metal ions and is strongly stimulated by the presence of the viral DNA replication origins. By means of this reaction, in which the TP is acting as a primer, Graphic29 DNA polymerase catalyzes the initiation step of Graphic29 DNA replication(5, 8). In addition to the synthetic activities, Graphic29 DNA polymerase has two degradative activities: 1) pyrophosphorolysis, the polymerization reversal, whose physiological significance is still unclear(10); 2) 3′-5′-exonuclease, shown to be involved in a proofreading function(11, 12). This activity, kinetically characterized using ssDNA as substrate and MgGraphic as metal activator(13), degrades processively DNA substrates longer than six nucleotides, the catalytic constant being 500 sGraphic. When the DNA length is reduced below 6-4 nucleotides, the Graphic29 DNA polymerase-ssDNA complex dissociates at a rate of 1 sGraphic. The multiple enzymatic activities of Graphic29 DNA polymerase (summarized in Table 1) allow this enzyme to be the only polymerase involved in the replication of the Graphic29 genome(7, 14). Moreover, the enzyme has two intrinsic properties: high processivity (>70 kilobases) and strand displacement ability(15). Based on this enzymatic potential, complete replication of both DNA strands can proceed continuously from each terminal priming event, without the need of synthesis of RNA-primed Okazaki fragments and making unnecessary the participation of accessory proteins and DNA helicases. The efficiency of the protein-primed initiation reaction is in part guaranteed by the previous formation of a heterodimer between TP and DNA polymerase(16), whereas the nucleotide specificity, as in normal DNA polymerization, is dictated by the DNA template(17).

This minireview will be reprinted in the 1995 Minireview Compendium, which will be available in 12, 1995. This investigation has been aided by Research Grant 5R01 GM27242-16 from the National Institutes of Health, by Grant PB93-0173 from Dirección General de Investigación Científica y Técnica, by Grant CHRX-CT 93-0248 from the European Economic Community, and by an institutional grant from Fundación Ramón Areces

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