DNA-protein interactions are central to the life cycle of all of the Herpes viruses. Herpes Simplex type 1 ICP8 protein is a delayed early protein that binds DNA and has been implicated in multiple stages of HSV DNA metabolism. It was recently discovered in this laboratory that ICP8 will catalyze DNA strand transfer reactions. Lehman and colleagues have shown that the HSV origin binding protein, UL-9, has a potent helicase activity in the presence of ICP8, and that it forms a heterodimer with ICP8 consisting of one monomer of ICP8 and one monomer of UL-9. In this project a major focus will be on further characterizing the interactions of ICP8, UL-9, an the human single strand binding protein, RPA in their catalysis of basic DNA transactions. A major tool of this study will be electron microscopy (EM). Previous preparations of ICP8 contained a small amount of residual exonuclease activity. Here ample quantities of ICP8 will be purified from HSV-infected cells to a level where the nuclease can either be eliminated totally or shown to be an integral part of ICP8. RPA will be purified from human cells and UL-9 will be obtained via collaboration. Alternatively new Baculovirus preparations of ICP8 may provide active material. In the best characterized systems, a single strand DNA binding protein is required as a co-factor for the strand transferases. RPA binds single strand DNA (ssDNA) tightly and HSV proteins will encounter RPA in the ell. Interactions between ICP8 and phosphorylated forms of RPA will be examined to determine if RPA will stimulate ICP8 or inhibit its activities. In a continuing collaboration the interaction of purified UL-9 protein with the HSV origin (ori-s) will be examined. This EM study will focus on binding sites, how the DNA is arranged by UL-9, whether RPA or ICP8 will facilitate the opening of the origin by UL-9, and finally how ICP8 promotes the helicase activity of UL-9. Much work is needed to further characterize the strand transfer properties of iCP8. This will employ ICP8, RPA, and UL-9 and use EM and biochemical assays. The preference of ICP8 for different DNA templates, whether it will catalyze exchange on nucleosomal DNA, and whether it will catalyze exchange at nicks or gaps in DNA will be examined. DNA templates will be designed to test the hypothesis that iCP8 and UL-9 can create structures that will prime secondary rounds of DNA replication.
Showing the most recent 10 out of 324 publications
