Helicase assembly protein Gp59 of bacteriophage T4: Fluorescence anisotropy and sedimentation studies of complexes formed with derivatives of Gp32, the phage ssDNA binding protein

The gene 59 protein (gp59) of bacteriophage T4 performs a vital function in phage DNA replication by directing the assembly of gp41, the DNA helicase component of the T4 primosome, onto lagging strand ssDNA at nascent replica tion forks. The helicase assembly activity of gp59 is required for optimum efficiency of helicase acquisition by the replication fork during strand di splacement DNA synthesis and is essential for helicase and primosome assemb ly during T4 recombination-dependent DNA replication transactions. Of centr al importance is the ability of gp59 to load the gp41 helicase onto ssDNA p reviously coated with cooperatively bound molecules of gp32, the T4 ssDNA b inding protein. Gp59 heteroassociations with ssDNA, gp32, and gp41 all appe ar to be essential for this loading reaction. Previous studies demonstrated that a tripartite complex containing gp59 and gp32 simultaneously cooccupy ing ssDNA is an essential intermediate in gp59-dependent helicase loading; however, the biochemical and structural parameters of gp59-gp32 complexes w ith or without ssDNA are currently unknown. To better understand gp59-gp32 interactions, we performed fluorescence anisotropy and analytical ultracent rifugation experiments employing native or rhodamine-labeled gp59 species i n combination with altered forms of gp32, allowing us to determine their bi nding parameters, shape parameters, and other hydrodynamic properties. Two truncated forms of gp32 were used: gp32-B, which lacks the N-terminal B-dom ain required for cooperative binding to ssDNA and for stable self-associati on, and A-domain fragment, which is the C-terminal peptide of gp32 lacking ssDNA binding ability. Results indicate that gp59 binds with high affinity to either gp32 derivative to form a 1:1 heterodimer. In both cases, heterod imer formation is accompanied by a conformational change in gp59 which corr elates with decreased gp59-DNA binding affinity. Hydrodynamic modeling sugg ests an asymmetric prolate ellipsoid shape for gp59, consistent with its X- ray crystallographic structure, and this asymmetry appears to increase upon binding of gp32 derivatives. Implications of our findings for the structur e and function of gp59 and gp59-gp32 complexes in T4 replication are discus sed.