Single Molecule Studies of Bacteriophage phi29 DNA Terminal Protein Interactions and their Role in DNA Packaging

Abstract

The assembly of a functional DNA-packaging complex is an essential process in the lifecycle of the tailed double-stranded DNA bacteriophage, phi29; however a detailed understanding of the assembly pathway is still missing. Phi29 and its relatives, as well as some Adenoviruses, possess linear double stranded DNA genomes with proteins covalently linked to the 5’ terminal ends of each strand. In addition to the terminal protein's (TP) main role in protein-primed DNA replication, the phi29 TP, gp-3, appears to play an essential role in the formation of the mature DNA packaging substrate. Evidence suggests gp3 mediates the formation of DNA loops which are subsequently bound by the motor protein, gp16, and supercoiled to form the mature packaging substrate. We have developed a single molecule optical tweezers assay that allows us to measure gp3-DNA and gp3-gp3 interactions and probe their role in the initiation of motor-driven viral DNA packaging. Here we present the direct observation of strong sequence-specific gp3-phi29DNA interactions as well as evidence for preferential binding of left-end gp3 to a region near the left terminal end of the genome, with the later suggesting a mechanism by which the virus may preferentially package the left end of its genome in vivo. We will also discuss efforts to determine whether gp16 in isolation, apart from the viral capsid portal complex, can act as a DNA translocase.