Identification of a Region of the Herpes Simplex Virus Single-Stranded DNA-Binding Protein Involved in Cooperative Binding

Abstract

We have identified a region of the herpes simplex virus major DNA-binding protein (ICP8) which is involved in cooperative binding to single-stranded DNA. This has been accomplished by analysis of ICP8 which was covalently modified by reaction with the extrinsic fluorophore fluorescein-5-maleimide (FM). Reaction conditions which result in the incorporation of 1 mol of FM per mol of ICP8 have been established. The binding properties of the modified protein were analyzed by polyacrylamide gel shift analysis with model oligonucleotides. This analysis indicates that while intrinsic binding is similar to that observed with unmodified protein, the cooperative binding of the modified protein to single-stranded DNA is significantly altered. Helix-destabilizing assays, whose results are a reflection of cooperative binding, also indicate that this property of ICP8 is decreased upon modification with FM. Mapping of the site of modification by cyanogen bromide cleavage and peptide sequencing has shown that the major site of modification is cysteine 254. This position in the primary structure of ICP8 is distinct from the regions previously shown to be involved in the interaction of this protein with single-stranded DNA.