Characterization of a herpes simplex virus type 2 deoxyuridine triphosphate nucleotidohydrolase and mapping of a gene conferring type specificity for the enzyme.
The herpes simplex virus type 2 (HSV-2)-induced deoxyuridine triphosphate nucleotidohydrolase (dUTPase) was purified approximately 600 +/- 43-fold using a combination of affinity, hydrophobic, absorption, and ion-exchange chromatography techniques. The only substrate for the dUTPase was dUTP with a Km of 3.6 +/- 1.1 microM. There was no apparent divalent cation requirement, but the HSV-2-induced dUTPase was inhibited by EDTA (0.1 mM) and this inhibition was reversed by either Co2+ (0.5 mM) or Mg2+ (0.5 mM). The HSV-2-induced dUTPase was distinguished from the HSV-1-induced and cellular dUTPases based upon differences in sensitivity to substrate inhibition, thermostability, and electrophoretic migration in nondenaturing polyacrylamide gels. Analysis of HSV-1 temperature-sensitive (ts) mutants demonstrated that ts A15 and ts K13 did not induce significant amounts of dUTPase activity at the permissive or nonpermissive temperatures. Mutants with defects in HSV-induced DNA polymerase or in the major DNA binding protein induced dUTPase at both temperatures. In contrast ts mutants defective in the alpha polypeptide VP175 (ICP4) did not induce normal levels of dUTPase at the nonpermissive temperature. The location of a gene encoding for the type specificity of the HSV induced dUTPase was mapped to the left 20% of the genome in Us in the region 0.060 to 0.100 or from 0.148 to 0.204.
D002413:Cations, Divalent; D002874:Chromosome Mapping; D004591:Electrophoresis, Polyacrylamide Gel; D005814:Genes, Viral; D009154:Mutation; D011489:Protein Denaturation; D011755:Pyrophosphatases / Q000235:genetics / Q000378:metabolism*; D018139:Simplexvirus / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
D002413:Cations, Divalent; D011755:Pyrophosphatases; C020020:dUTP pyrophosphatase