Replication of bacteriophage phi 29 DNA in vitro: the roles of terminal protein and DNA polymerase.

Abstract

Phage .vphi.29 DNA replication is initiated by the formation of a covalent complex between the viral-coded terminal protein and dAMP (TP-dAMP). This initiation reaction system was reconstituted from 2 phage-encoded proteins, the terminal protein and DNA polymerase. The .vphi.29 DNA polymerase was purified from phage-infected cells by using poly(dA).cntdot.p(dT)12-18 as an assay template. The purified polymerase has an apparent MW of 68 kDa [kilodalton] in its native form and it appears to function as a monomer. The terminal protein was purified to homogeneity from Escherichia coli cells harboring a cloned plasmid that contained a .vphi.29 gene 3 segment. The MW of the purified terminal protein was .apprx. 30 kDa in both the denatured and the native form. The protein apparently functions as a monomer. When the terminal protein and DNA polymerase was incubated in the presence of dATP, Mg2+ and .vphi.29 DNA-protein as template, the terminal protein bound covalently to dAMP. This reaction did not require ATP. These 2 purified fractions catalyzed DNA chain elongation from both ends of .vphi.29 DNA, yielding the expected 9-12-base fragment when assayed in the presence of 2'',3''-dideoxycytidine triphosphate. Evidently, .vphi.29 DNA polymerase catalyzes formation of the terminal protein-dAMP complex and can also catalyze chain elongation at least 9-12 bases from both ends of .vphi.29 DNA.