The herpes simplex virus type I origin binding protein. DNA-dependent nucleoside triphosphatase activity.
A recombinant baculovirus overexpressing the herpes simplex virus type 1 (HSV-1) origin binding protein, encoded by the UL9 gene, was constructed. The purified recombinant protein has DNA-dependent nucleoside triphosphatase activity similar to the enzyme isolated from mammalian cells. Optimal nucleoside triphosphatase activity requires low salt (< 50 mM), 2-3 mM Mg2+, alkaline pH (8.3-9.5), high temperature (45 degrees C), and a single-stranded DNA coeffector containing minimal secondary structure. Enzymatic activity is subject to product inhibition, and there appears to be a single nucleotide binding site. The minimal length of single-stranded DNA that elicits enzymatic activity is 14 nucleotides, and activity increases as the length is increased. Saturation for various single-stranded DNA coeffectors is about 10 microM in nucleotide, but the maximum velocity is reduced 2-3-fold for coeffectors containing secondary structure. The HSV-1-encoded single-stranded DNA-binding protein ICP8 specifically stimulates the DNA-dependent nucleoside triphosphatase activity. The kinetics of nucleoside triphosphate hydrolysis exhibit a substantial lag period which can be shortened, but not eliminated, by reduced secondary structure in the DNA coeffector or by increased temperature.
Department of Biochemistry, Beckman Center, Stanford University School of Medicine, California 94305-5307.
M S
MS
Dodson
I R
IR
Lehman
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D004265:DNA Helicases; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005814:Genes, Viral; D006863:Hydrogen-Ion Concentration; D007313:Insecta; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D011994:Recombinant Proteins / Q000378:metabolism; D012265:Ribonucleotides / Q000378:metabolism; D018139:Simplexvirus / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity; D013816:Thermodynamics; D014162:Transfection; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D012265:Ribonucleotides; D014764:Viral Proteins; C064565:UL9 protein, Human herpesvirus 1; D000251:Adenosine Triphosphatases; D004265:DNA Helicases; D008274:Magnesium