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J Biol Chem, Vol. 275, Issue 15, 10864-10869, April 14, 2000
From the 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° C, K = 0.55 ± 0.05 × 106 M
Equilibrium Binding of Single-stranded DNA with Herpes Simplex
Virus Type I-coded Single-stranded DNA-binding Protein, ICP8*
,,,¶
Institut de Pharmacologie et de Biologie
Structurale, CNRS, 205 Route de Narbonne, 31077 Toulouse
Cédex, France and § Department of Biochemistry and
Molecular Biology, University of Miami School of Medicine,
Miami, Florida 33101-6129
1, and a cooperativity
parameter, 
= 15 ± 3. The equilibrium constant was
largely independent of salt,
log(K)/log([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.
*
This work was supported in part by Grants 9627 and 9584 from
the Association pour la Recherche sur le Cancer (to N. P. J. and
G. V., respectively) and by Grant AI38335 from the National Institutes
of Health (to P. E. B.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
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