[5] Purification of bacteriophage φ29 DNA polymerase

This chapter describes the protocols for the preparation of φ29 DNA polymerase in a highly purified form. These methods represent significant improvements at both the overproduction and the purification levels. Bacteriophage φ29 DNA polymerase, the product of the viral gene, was originally characterized as a protein involved in the initiation of φ29 DNA replication based on both in vivo and in vitro studies. The two distinguishable synthetic reactions catalyzed by this enzyme are (1) DNA polymerization, as any other DNA-dependent DNA polymerase, the template-directed addition of dNMP units from dNTPs. It occurs on a DNA or RNA primer strand, in the presence of divalent metal ions and (2) Terminal protein (TP) deoxynucleotidylation, which is the formation of a covalent linkage (phosphoester) between the hydroxyl group of a specific serine residue (Ser-232) in φ29 TP and 5'-dNMP. It uses any of the four dNTPs as substrate, in the presence of divalent metal ions. Two degradative activities of φ29 DNA polymerase are (1) pyrophosphorolysis and (2) 3' ∼ 5'-exonuclease or metal-dependent excision of dNMPs.