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 ICPS is a 128-kDa zinc metalloprotein, In this communication we have shown that unsu bstituted and bromodeoxyuridine-substituted oligonucleotides can be specifi cally 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 residu es 368-902 synthesized in vibro exhibited single-strand DNA-binding activit y, We conclude that the region encompassing residues 368-902 contains the s ingle-strand DNA-binding site of ICP8. Moreover, photoaffinity labeling of ICP8 with oligonucleotides provides a means of specifically modifying its s ingle-strand DNA-binding site, thereby facilitating future studies on the i mportance of its single-strand DNA-binding activity in its interaction with other DNA replication enzymes. (C) 1999 Academic Press.