Simultaneous interactions of bacteriophage T4 DNA replication proteins gp59 and gp32 with single-stranded (ss) DNA - Co-modulation of ssDNA binding activities in a DNA helicase assembly intermediate

The T4 gp59 protein is the major accessory protein of the phage's replicati ve DNA helicase, gp41. gp59 helps load gp41 at DNA replication forks by pro moting its assembly onto single-stranded (ss) DNA covered with cooperativel y bound molecules of gp32, the T4 single-strand DNA binding protein (ssb). A gp59-gp32-ssDNA ternary complex is an obligatory intermediate in this hel icase loading mechanism. Here, we characterize the properties of gp59-gp32- ssDNA complexes and reveal some of the biochemical interactions that occur within them. Our results indicate the following: (i) gp59 is able to co-occ upy ssDNA pre-saturated with either gp32 or gp32-A (a truncated gp32 specie s lacking interactions with gp59); (ii) gp59 destabilizes both gp32-ssDNA a nd (gp32-A)-ssDNA interactions; (iii) interactions of gp59 with the A-domai n of gp32 alter the ssDNA-binding properties of gp59; and (iv) gp59 organiz es gp32-ssDNA versus (gp32-A)-ssDNA into morphologically distinct complexes . Our results support a model in which gp59-gp32 interactions are non-essen tial for the co-occupancy of both proteins on ssDNA but are essential for t he formation of structures competent for helicase assembly. The data argue that specific "cross-talk" between gp59 and gp32, involving conformational changes in both, is a key feature of the gp41 helicase assembly pathway.