ABSTRACT
In this chapter the authors provide a description of the techniques their laboratory has found useful for studying the two subunits of the herpes simplex virus DNA polymerase. They describe a transient genetic assay that allows phenotypic assessment of the mutants in the context of an infected cell, permitting a correlation between the in vitro biochemical activities of a protein and its function in vivo. Herpes simplex virus provides an excellent model system for the study of DNA replication in eukaryotic cells, in part because it encodes many, if not all, of the proteins that are directly required for replication of the virus genome. The virus is also a ubiquitous human pathogen whose control relies primarily on antiviral drugs, the most successful of which are directed against the catalytic site of the DNA polymerase. Analysis of in vitro translated non-sequence-specific DNA binding proteins is complicated by the large quantities of endogenous nucleic acid binding proteins present in rabbit reticulocyte lysate.
