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 alpha-like DNA polymerase and also an antiviral drug target. To investigate its organization, we mapped favored cleavage sites for seven proteases and identified three major classes of stable proteolytic fragments: 70-85-kDa N-terminal fragments, 50-70-kDa fragments that start near residues 600-700, and 12-kDa C-terminal fragments. In coimmunoprecipitation experiments, the first two classes of fragments remained associated; thus, cleavage in the center of the protein did not resolve structurally separate domains. In contrast, the 12-kDa C-terminal fragments did not remain associated with other fragments, suggesting a small separable C-terminal domain. The 70-85-kDa N-terminal fragments contained 3'-5' exonuclease and ribonuclease H activities; however, cleavage at the center of the molecule or near the C terminus appeared to destroy DNA polymerase activity. All three major classes of fragments bound DNA in DNA-cellulose chromatography and Southwestern blot analyses. The 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 pertinent to other DNA polymerases.
Weisshart
K
K
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Kuo
A A
AA
Hwang
C B
CB
Kumura
K
K
Coen
D M
DM
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000378:metabolism; D018259:Herpesvirus 1, Human / Q000201:enzymology*; D015151:Immunoblotting; D007313:Insecta; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D010449:Peptide Mapping; D016914:Ribonuclease H / Q000378:metabolism; D014162:Transfection; D014764:Viral Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism
D010446:Peptide Fragments; D014764:Viral Proteins; D004259:DNA-Directed DNA Polymerase; D005090:Exodeoxyribonucleases; C043417:DNA polymerase, Simplexvirus; D043211:Exodeoxyribonuclease V; D016914:Ribonuclease H