Protein-primed replication of bacteriophage Φ29 DNA

Abstract

The replication of Φ29 DNA-protein p3 represents a simple model system to study the protein-priming mechanism of initiation of replication. The Φ29 DNA polymerase involved both in the initiation and elongation steps of Φ29 DNA-protein p3 replication, is a very processive enzyme and it is able to produce strand-displacement in the absence of other proteins. To correlate functional and structural domains in the Φ29 DNA polymerase point mutants in the most carboxyl region of amino-acid homology with other DNA polymerases have been constructed. Most of the mutations had a decreased initiation and elongation activity, but normal 3′ → 5′ exonuclease activity, suggesting that this region contributes to the active domain for initiation and elongation. Point and deletion mutants in the terminal protein have allowed the mapping of one DNA-binding region and two DNA-polymerase-binding regions. The viral protein p6, which stimulates the initiation of replication, binds to a set of specific signals present at both Φ29 DNA ends. A good correlation of binding and stimulation of replication has been obtained by using fragments containing Φ29 DNA-terminal sequences and deletion mutants of protein p6. The viral protein p5 has been shown to bind to single-stranded DNA, to protect the latter against nuclease digetion, and to stimulate Φ29 DNA-protein p3 replication in vitro.