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

A S Gourves¹, N Tanguy Le Gac, G Villani

¹Institut de Pharmacologie et de Biologie Structurale, CNRS, 205 Route de Narbonne, 31077 Toulouse Cédex, France.

The Journal of Biological Chemistry

|February 7, 2001

Summary

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Herpes simplex virus type I

Area of Science:

Molecular Biology
Virology
Biochemistry

Background:

ICP8 is the primary single-stranded DNA (ssDNA)-binding protein in herpes simplex virus type I.
Understanding ICP8's DNA binding is crucial for comprehending viral replication and recombination mechanisms.

Purpose of the Study:

To determine the thermodynamic parameters of ICP8's interaction with ssDNA.
To elucidate the binding mechanism and compare it with other ssDNA-binding proteins.

Main Methods:

Solution equilibrium binding studies.
Fluorescence anisotropy measurements using a 5'-fluorescein-labeled 32-mer oligonucleotide.
Analysis of binding site size, association constant, and cooperativity.

Main Results:

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ICP8 forms a nucleoprotein filament on ssDNA with a binding site size of 10 nucleotides/ICP8 monomer.
The association constant (K) at 25°C is 0.55 ± 0.05 x 10^6 M⁻¹, with a cooperativity parameter (ω) of 15 ± 3.
ICP8 exhibits unusual binding characterized by low cooperativity and weak binding, with increased product stability at high salt concentrations.

Conclusions:

ICP8's binding mechanism is distinct from many other ssDNA-binding proteins.
ICP8 shares characteristics with recombinase active conformations, suggesting a potential similar role in catalyzing homologous recombination.
These findings provide insights into the function of ICP8 in viral DNA metabolism and recombination.