Dissecting the role of the 29 terminal protein DNA binding residues in viral DNA replication

Phage 29 DNA replication takes place by a protein-priming mechanism in which the viral DNA poly-merase catalyses the covalent linkage of the initi-ating nucleotide to a specific serine residue of the terminal protein (TP). The N-terminal domain of the 29 TP has been shown to bind to the host DNA in a sequence-independent manner and this binding is essential for the TP nucleoid localisation and for an efficient viral DNA replication in vivo. In the present work we have studied the involvement of the TP N-terminal domain residues responsible for DNA bind-ing in the different stages of viral DNA replication by assaying the in vitro activity of purified TP N-terminal mutant proteins. The results show that mutation of TP residues involved in DNA binding affects the catalytic activity of the DNA polymerase in initiation, as the Km for the initiating nucleotide is increased when these mutant proteins are used as primers. Importantly, this initiation defect was relieved by using the 29 double-stranded DNA binding protein p6 in the reac-tion, which decreased the Km of the DNA polymerase for dATP about 130–190 fold. Furthermore, the TP N-terminal domain was shown to be required both for a proper interaction with the DNA polymerase and for an efficient viral DNA amplification