CHARACTERIZATION OF AN AMINO-TERMINAL FRAGMENT OF THE BACTERIOPHAGE-T4 UVSY RECOMBINATION PROTEIN

The uvsY protein plays essential roles in homologous genetic recombina tion processes in the bacteriophage T4. In vitro, uvsY promotes the fo rmation of presynaptic filaments containing stoichiometric amounts of the T4 uvsX recombinase bound to single-stranded DNA. uvsY protein has intrinsic binding activities towards ssDNA, uvsX, and gp32, the T4-en coded SSB, however, it has not been directly determined which of these activities are essential for uvsY's role in presynapsis. We have ther efore sought to generate altered forms of uvsY deficient in uvsX- and/ or gp32-binding, in order to assess whether these specific protein-pro tein interactions are essential for uvsY recombination functions. Limi ted chymotrypsinolysis of the 16 kDa uvsY protein generates two major fragments: an 11.5 kDa fragment containing the N-terminus of uvsY, and a 4.5 kDa C-terminal fragment. We have expressed and purified the lar ge fragment as a fusion protein containing the N-terminal 101 amino ac ids of uvsY. We show that this truncated uvsY species, which we call U VSYNT, retains ssDNA-binding activity, but is devoid of both uvsX- and gp32-binding activities. Like native uvsY, uvsY(NT) stimulates the ss DNA-dependent ATPase activity of the uvsX protein, however, the synerg istic effects observed between uvsY, uvsX, and gp32 are not observed w ith uvsY(NT). in addition, uvsY(NT) weakly stimulates uvsX-catalyzed D NA strand exchange reactions. The latter result is surprising since it suggests that specific interactions with uvsX and/or gp32 are not abs olutely essential for uvsY recombination functions. Taken together, th e data are consistent with a model in which uvsY-ssDNA interactions al one are capable of promoting the assembly of functional uvsX-ssDNA com plexes, while uvsY-protein interactions stabilize uvsX-ssDNA complexes .