THE GENE-59 PROTEIN OF BACTERIOPHAGE-T4 - CHARACTERIZATION OF PROTEIN-PROTEIN INTERACTIONS WITH GENE-32 PROTEIN, THE T4 SINGLE-STRANDED-DNABINDING-PROTEIN

The gene 59 protein (gp59) of bacteriophage T4 stimulates the activiti es of gene 41 protein (gp41), the T4 replicative DNA helicase, by prom oting the assembly of gp41 onto single-stranded (ss)-DNA molecules tha t are covered with cooperatively bound gene 32 protein (gp32). This he licase-ssDNA assembly process, which is important for the reconstituti on of the primosome component of the T4 DNA replication fork, appears to require both gp59-gp41 and gp59-gp32 protein-protein interactions. In this study we characterize the physical and functional interactions of gp59 with gp32, the T4 ssDNA-binding protein. Experimental results presented herein indicate: 1) that gp59 binds specifically to both fr ee and ssDNA-bound gp32 molecules; and 2) that in both cases binding i nvolves contacts of gp32 (the so-called ''A-domain''). We further show that single-stranded DNA molecules coated with (gp32-A), a truncated form of gp32 lacking A-domain, are refractory to gp59-dependent helica se assembly. The data indicate that specific contacts between gp59 mol ecules and the A-domains of gp32 molecules are essential for gp59-depe ndent assembly of gp41 onto gp32-ssDNA complexes. Our results are cons istent with a model in which gp59 binds to gp32 molecules within the g p32-ssDNA complex and therein forms a target site for helicase-ssDNA a ssembly.