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 with ssDNA. Fluorescence anisotropy measurements of a 5'-fluorescein-labeled 32-mer oligonucleotide revealed that ICP8 formed a nucleoprotein filament on ssDNA with a binding site size of 10 nucleotides/ICP8 monomer, an association constant at 25 degrees C, K = 0.55 +/- 0.05 x 10(6) M(-1), and a cooperativity parameter, omega = 15 +/- 3. The equilibrium constant was largely independent of salt, deltalog(Komega)/deltalog([NaCl]) = -2.4 +/- 0.4. Comparison of these parameters with other ssDNA-binding proteins showed that ICP8 reacted with an unusual mechanism characterized by low cooperativity and weak binding. In addition, the reaction product was more stable at high salt concentrations, and fluorescence enhancement of etheno-ssDNA by ICP8 was higher than for other ssDNA-binding proteins. These last two characteristics are also found for protein-DNA complexes formed by recombinases in their active conformation. Given the proposed role of ICP8 in promoting strand transfer reactions, they suggest that ICP8 and recombinase proteins may catalyze homologous recombination by a similar mechanism.
Gourves
A S
AS
Institut de Pharmacologie et de Biologie Structurale, CNRS, 205 Route de Narbonne, 31077 Toulouse Cédex, France.
Tanguy Le Gac
N
N
Villani
G
G
Boehmer
P E
PE
Johnson
N P
NP
D004277:DNA, Single-Stranded / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D009841:Oligonucleotides / Q000378:metabolism; D012965:Sodium Chloride / Q000494:pharmacology; D013696:Temperature; D014764:Viral Proteins / Q000378:metabolism*
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; C045462:ICP8 protein, Simplexvirus; D009841:Oligonucleotides; D014764:Viral Proteins; D012965:Sodium Chloride