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 large catalytic (UL30 gene product) and a small auxiliary subunit (UL42 gene product). The DNA binding of HSV pol, its cofactor, and the assembled holoenzyme complex was studied by bandshift analysis using purified proteins expressed via recombinant baculovirus. The functional activity of the recombinant UL42, purified by phenyl-Sepharose chromatography, was confirmed by its ability (1) to convert the salt sensitivity of both, 3'-5' exonuclease and polymerase, activities of HSV pol and (2) to enhance the processivity of polymerization. Bandshift analyses revealed that the HSV pol holoenzyme-DNA complex was stably formed up to a salt concentration of 50 mM ammonium sulfate, indicating that the restricted DNA and protein interactions of both HSV pol and UL42 are responsible for the observed salt preference of the HSV pol holoenzyme under standard assay conditions in vitro. Studies of the assembly of the holoenzyme complex demonstrated that initial DNA binding of HSV pol was advantageous for the formation of the HSV pol holoenzyme-DNA complex.
Franz
C
C
Division of Clinical Chemistry and Biochemistry, University of Zurich, Steinwiesstrasse 75, Zurich, CH-8032, Switzerland.
Kühn
F J
FJ
Knopf
C W
CW
D000818:Animals; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005090:Exodeoxyribonucleases / Q000378:metabolism*; D018259:Herpesvirus 1, Human / Q000201:enzymology*; D007106:Immune Sera; D015183:Restriction Mapping; D018411:Spodoptera; D014162:Transfection; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D004279:DNA, Viral; D007106:Immune Sera; D014764:Viral Proteins; D004259:DNA-Directed DNA Polymerase; D005090:Exodeoxyribonucleases; C043417:DNA polymerase, Simplexvirus