STRUCTURAL AND FUNCTIONAL-ORGANIZATION OF HERPES-SIMPLEX VIRUS-DNA POLYMERASE INVESTIGATED BY LIMITED PROTEOLYSIS

The 1235 residue herpes simplex virus DNA polymerase is a prototype al pha-like DNA polymerase and also an antiviral drug target. To investig ate its organization, we mapped favored cleavage sites for seven prote ases and identified three major classes of stable proteolytic fragment s: 70-85-kDa N-terminal fragments, 50-70-kDa fragments that start near residues 600-700, and 12-kDa C-terminal fragments. In coimmunoprecipi tation experiments, the first two classes of fragments remained associ ated; thus, cleavage in the center of the protein did not resolve stru cturally separate domains. In contrast, the 12-kDa C-terminal fragment s did not remain associated with other fragments, suggesting a small s eparable C-terminal domain. The 70-85-kDa N-terminal fragments contain ed 3'-5' exonuclease and ribonuclease H activities; however, cleavage at the center of the molecule or near the C terminus appeared to destr oy DNA polymerase activity. All three major classes of fragments bound DNA in DNA-cellulose chromatography and Southwestern blot analyses. T he C-terminal fragments bound the viral polymerase processivity factor , UL42. The results map activities to regions of herpes simplex virus polymerase and suggest a model for its organization that may be pertin ent to other DNA polymerases.