Bacteriophage ϕ29 DNA Polymerase

The most peculiar characteristic of the linear DNA molecule (19285 bp) of bacteriophage ϕ29 is the presence of a terminal protein (TP) covalently bound to both 5′ ends. The elucidation of the mechanism of ϕ29 DNA replication, together with the characterization of the proteins involved in the different stages of this process, indicated that ϕ29 TP acts as a protein primer for the initial step of ϕ29 DNA replication (reviewed in Salas 1991). Thus, after formation of a TP-dAMP complex at both DNA ends, elongation proceeds by a strand displacement mechanism to complete replication of both ϕ29 DNA strands. As will be described in this chapter, the enzymology of ϕ29 DNA replication is mainly based on the multiple catalytic activities and peculiar properties of the ϕ29 gene 2 product: the ϕ29 DNA polymerase. This enzyme, with a molecular weight of only about 66 kDa, is the only polymerase involved in ϕ29 DNA replication, catalyzing both the initiation and elongation stages of DNA synthesis (Blanco and Salas 1984, 1985a). Moreover, the peculiar polymerization properties of ϕ29 DNA polymerase (high processivity and strand displacement) make the participation of other enzymatic activities or accessory functions unnecessary to improve its efficiency in DNA synthesis (Blanco et al. 1989). From the enzymatic point of view, ϕ29 DNA polymerase is able to catalyze two distinguishable synthetic reactions: TP-deoxynucleotidylation and DNA polymerization, and also two degradative reactions: pyrophosphorolysis and 3′–5′ exonucleolysis. These multiple catalytic activities, their associated properties (summarized in Table 1), and their structural mapping, will be described in the following sections.

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