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Biochemistry. 1999 Dec 14;38(50):16589-98.

Relationship between hexamerization and ssDNA binding affinity in the uvsY recombination protein of bacteriophage T4.

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Department of Biochemistry, Vermont Cancer Center, University of Vermont College of Medicine, Burlington 05405, USA.


In bacteriophage T4, homologous genetic recombination events are catalyzed by a presynaptic filament 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 bound gp32 (the T4 ssDNA-binding protein) from the ssDNA, a thermodynamically unfavorable reaction. This displacement is mediated by the T4 uvsY protein (15.8 kDa, 137 amino acids), which interacts with both uvsX- and gp32-ssDNA complexes and modulates their properties. Previously, we showed that uvsY exists as a hexamer under physiological conditions and that uvsY hexamers bind noncooperatively but with high affinity to ssDNA. We also showed that a fusion protein containing the N-terminal 101 amino acid residues of uvsY lacks interactions with uvsX and gp32 but retains both weak ssDNA-binding activity and a residual ability to stimulate uvsX-catalyzed recombination functions. Here, we present quantitative data on the oligomeric structure and ssDNA-binding properties of a closely related fusion protein designated uvsY. Sedimentation velocity and equilibrium results establish that uvsY, unlike native uvsY, behaves as a monomer in solution (M(app) = 14.2 kDa, = 2.1). Like native uvsY, uvsY binds noncooperatively to an etheno-DNA (epsilonDNA) lattice with a binding site size of 4 nucleotides/monomer; however at physiological ionic strength, the association constant for uvsY-epsilonDNA is decreased 10(4)-fold relative to native uvsY. Nevertheless, the magnitude of the salt effect on the association constant (K) is essentially unchanged between uvsY and uvsY, indicating that disruption of the C-terminus does not disrupt the electrostatic ssDNA-binding determinants found within each protomer of uvsY. Instead, the large difference in ssDNA-binding affinities reflects the loss of hexamerization ability by uvsY, suggesting that a form of intrahexamer synergism or cooperativity between binding sites within the uvsY hexamer leads to its high observed affinity for ssDNA.

[Indexed for MEDLINE]

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