Photoaffinity labeling of the herpes simplex virus type-1 single-strand DNA-binding protein (ICP8) with oligodeoxyribonucleotides.

The herpes simplex virus type-1 single-strand DNA-binding protein ICP8 is a 128-kDa zinc metalloprotein. In this communication we have shown that unsubstituted and bromodeoxyuridine-substituted oligonucleotides can be specifically crosslinked to ICP8 by UV irradiation. We have used this approach to show that the single-strand DNA-binding site of ICP8 resides within a 53.5-kDa tryptic polypeptide. This polypeptide initiates at alanine 368 and was estimated to extend through arginine 902. A polypeptide encompassing residues 368-902 synthesized in vitro exhibited single-strand DNA-binding activity. We conclude that the region encompassing residues 368-902 contains the single-strand DNA-binding site of ICP8. Moreover, photoaffinity labeling of ICP8 with oligonucleotides provides a means of specifically modifying its single-strand DNA-binding site, thereby facilitating future studies on the importance of its single-strand DNA-binding activity in its interaction with other DNA replication enzymes.