| Abstract
| - In bacteriophage T4, homologous genetic recombination events are catalyzed by a presynapticfilament containing stoichiometric quantities of the T4 uvsX recombinase bound cooperatively to single-stranded DNA (ssDNA). The formation of this filament requires the displacement of cooperatively boundgp32 (the T4 ssDNA-binding protein) from the ssDNA, a thermodynamically unfavorable reaction. Thisdisplacement is mediated by the T4 uvsY protein (15.8 kDa, 137 amino acids), which interacts with bothuvsX− and gp32−ssDNA complexes and modulates their properties. Previously, we showed that uvsYexists as a hexamer under physiological conditions and that uvsY hexamers bind noncooperatively butwith high affinity to ssDNA. We also showed that a fusion protein containing the N-terminal 101 aminoacid residues of uvsY lacks interactions with uvsX and gp32 but retains both weak ssDNA-binding activityand a residual ability to stimulate uvsX-catalyzed recombination functions. Here, we present quantitativedata on the oligomeric structure and ssDNA-binding properties of a closely related fusion protein designateduvsY*. Sedimentation velocity and equilibrium results establish that uvsY*, unlike native uvsY, behavesas a monomer in solution (Mapp = 14.2 kDa,= 2.1). Like native uvsY, uvsY* binds noncooperatively to an etheno-DNA (εDNA) lattice with a binding site size of 4 nucleotides/monomer; however atphysiological ionic strength, the association constant for uvsY*−εDNA is decreased 104-fold relative tonative uvsY. Nevertheless, the magnitude of the salt effect on the association constant (K) is essentiallyunchanged between uvsY and uvsY*, indicating that disruption of the C-terminus does not disrupt theelectrostatic ssDNA-binding determinants found within each protomer of uvsY. Instead, the large differencein ssDNA-binding affinities reflects the loss of hexamerization ability by uvsY*, suggesting that a formof intrahexamer synergism or cooperativity between binding sites within the uvsY hexamer leads to itshigh observed affinity for ssDNA.
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