Equilibrium binding of single-stranded DNA with herpes simplex virus type I-coded single-stranded DNA-binding protein, ICP8

We have carried out solution equilibrium binding studies of ICP8, the major single-stranded DNA (ssDNA)-binding protein of herpes simplex virus type I , in order to determine the thermodynamic parameters for its interaction wi th ssDNA, Fluorescence anisotropy measurements of a 5'-fluorescein-labeled 32-mer oligonucleotide revealed that ICP8 formed a nucleoprotein filament o n ssDNA with a binding site size of 10 nucleotides/ICP8 monomer, an associa tion constant at 25 degrees C, K = 0.55 +/- 0.05 x 10(6) M-1, and a coopera tivity parameter, omega = 15 +/- 3, The equilibrium constant was largely in dependent of salt, delta log(K omega)/delta log([NaCl]) = -2.4 +/- 0.4. Com parison of these parameters with other ssDNA-binding proteins showed that I CP8 reacted with an unusual mechanism characterized by low cooperativity an d weak binding. In addition, the reaction product was more stable at high s alt concentrations, and fluorescence enhancement of etheno-ssDNA by ICP8 wa s higher than for other ssDNA-binding proteins. These last two characterist ics are also found for protein-DNA complexes formed by recombinases in thei r active conformation. Given the proposed role of ICP8 in promoting strand transfer reactions, they suggest that ICP8 and recombinase proteins may cat alyze homologous recombination by a similar mechanism.