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

Anne Sophie Gourves, Nicolas Tanguy Le Gac, Giuseppe Villani, Paul E. Boehmer, Neil P. Johnson

Research output: Contribution to journalArticle

27 Scopus citations

Abstract

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-met 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 x 106 M-1, and a cooperativity parameter, ω = 15 ± 3. The equilibrium constant was largely independent of salt, δkog(Kω)/δlog([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.

Original languageEnglish (US)
Pages (from-to)10864-10869
Number of pages6
JournalJournal of Biological Chemistry
Volume275
Issue number15
DOIs
StatePublished - Apr 14 2000
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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