The 60-residue C-terminal region of the single-stranded DNA binding protein of herpes simplex virus type 1 is required for cooperative DNA binding

ICP8 is the major single-stranded DNA (ssDNA) binding protein of the herpes simplex virus type 1 and is required for the onset and maintenance of vira l genomic replication. To identify regions responsible for the cooperative binding to ssDNA, several mutants of ICP8 have been characterized. Total re flection X-ray fluorescence experiments on the constructs confirmed the pre sence of one zinc atom per molecule. Comparative analysis of the mutants by electrophoretic mobility shift assays was done with oligonucleotides for w hich the number of bases is approximately that occluded by one protein mole cule. The analysis indicated that neither removal of the 60-amino-acid C-te rminal region nor Cys254Ser and Cys455Ser mutations qualitatively affect th e intrinsic DNA binding ability of ICP8. The C-terminal deletion mutants, h owever, exhibit a total loss of cooperativity on longer ssDNA stretches. Th is behavior is only slightly modulated by the two-cysteine substitution. Ci rcular dichroism experiments suggest a role for this C-terminal tail in pro tein stabilization as well as in intermolecular interactions. The results s how that the cooperative nature of the ssDNA binding of ICPS is localized i n the 60-residue C-terminal region. Since the anchoring of a C- or N-termin al ann of one protein onto the adjacent one on the DNA strand has been repo rted for other ssDNA binding proteins, this appears to be the general struc tural mechanism responsible for the cooperative ssDNA binding by this class of protein.