DNA and protein interactions of the small subunit of herpes simplex virus type 1 DNA polymerase

Herpes simplex virus DNA polymerase (HsV pol) holoenzyme consists of a larg e catalytic (UL30 gene product) and a small auxiliary subunit (UL42 gene pr oduct). The DNA binding of HSV pol, its cofactor, and the assembled holoenz yme complex was studied by bandshift analysis using purified proteins expre ssed via recombinant baculovirus. The functional activity of the recombinan t UL42, purified by phenyl-Sepharose chromatography, was confirmed by its a bility (1) to convert the salt sensitivity of both, 3'-5' exonuclease and p olymerase, activities of HsV pol and (2) to enhance the processivity of pol ymerization. Bandshift analyses revealed that the HSV pol holoenzyme-DNA co mplex was stably formed up to a salt concentration of 50 mM ammonium sulfat e, indicating that the restricted DNA and protein interactions of both HsV pol and UL42 are responsible for the observed salt preference of the HsV po l holoenzyme under standard assay conditions in vitro. Studies of the assem bly of the holoenzyme complex demonstrated that initial DNA binding of HSV pol was advantageous for the formation of the HSV pol holoenzyme-DNA comple x. (C) 1999 Academic Press.