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

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:

• 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.