Improved assays for DNA-polymerizing enzymes by the use of enzymatically synthesized 5-[125I]iodo-2'-deoxyuridine triphosphate, illustrated by direct quantitation of anti-HIV reverse transcriptase antibody and by serum DNA polymerase analyses.
A one-step procedure which uses enzymes in a crude extract of herpes simplex virus (HSV) type 1-infected cells to synthesize 5-[125I]iodo-2'-deoxyuridine triphosphate [( 125I]dUTP) from [125I]dU is described. The design of a one-step procedure for the purification of the product is also presented. The recovery of [125I]dUTP from [125I]dU varied between 50 and 75%, the radiochemical purity of the product was greater than 90%, and both synthesis and purification were completed within 8 h. The sensitivity and specificity of [125I]dUTP as a substrate for both DNA-dependent DNA polymerase (DNAp) and RNA-dependent DNA polymerase (reverse transcriptase, RT) were evaluated and compared to those of [3H]dTTP for the following specimens: purified cloned Klenow fragment, crude extracts of HeLa-, BHK-, and HSV-2-infected BHK cells, purified avian myeloblastosis virus RT, and purified cloned human immunodeficiency virus (HIV) RT. The [125I]dUTP was accepted as a substrate equally as well [3H]dTTP by all of the specimens at all of the concentrations tested. When the same amount of radiolabel was used, [125I]dUTP gave a sensitivity 10- to 25-fold higher than that of [3H]dTTP. The gain in sensitivity was due to the higher specific activity and a higher counting efficiency of the 125I-label compound. The use of [125I]dUTP also offered technical advantages over alternative substrates available, such as product separation without acid precipitation and exclusion of the need for scintillation cocktails. The half-life of the nucleic also gives a reasonable shelf-life for use in routine assays. Activity of less than 0.3 pg of HIV RT could be detected when the new substrate was used, and this made it possible to quantitate HIV RT antibodies (abs) in diluted serum samples without purifying the immunoglobulin. Analysis of 31 HIV-infected individuals showed that all of them had anti-HIV RT ab and that the amount of serum needed for 50% inhibition of the HIV RT activity corresponded to an amount of immunoglobulin 100-fold smaller (i.e., 0.02-31.4 micrograms) than has been previously reported. With the substrate it was also possible to detect DNAp activity in serum from healthy individuals, although a long-duration assay was required. In a long-duration assay the DNAp activity found in sera from healthy individuals was linear with respect to time, whereas the DNAp activity found in many sera from tumor patients was not. [125I]dUTP is judged to be an excellent substrate for detecting and quantifying the activity of various DNA-synthesizing enzymes and their blocking abs.
Research Unit of Replication Enzymology, Uppsala University, Sweden.
M
M
Neumüller
A R
AR
Karlström
C F
CF
Källander
J S
JS
Gronowitz
D000914:Antibodies, Viral / Q000032:analysis*; D002460:Cell Line; D004247:DNA / Q000096:biosynthesis; D004259:DNA-Directed DNA Polymerase / Q000032:analysis / Q000097:blood*; D003856:Deoxyuracil Nucleotides / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D006678:HIV / Q000201:enzymology*; D015658:HIV Infections / Q000276:immunology; D006367:HeLa Cells; D006801:Humans; D007457:Iodine Radioisotopes; D012194:RNA-Directed DNA Polymerase / Q000276:immunology* / Q000378:metabolism; D011994:Recombinant Proteins; D018139:Simplexvirus / Q000502:physiology; D013379:Substrate Specificity; D013942:Thymine Nucleotides / Q000378:metabolism
D000914:Antibodies, Viral; D003856:Deoxyuracil Nucleotides; D007457:Iodine Radioisotopes; D011994:Recombinant Proteins; D013942:Thymine Nucleotides; C008144:5-iodo-2'-deoxyuridine triphosphate; D004247:DNA; D012194:RNA-Directed DNA Polymerase; D004259:DNA-Directed DNA Polymerase; C024157:thymidine 5'-triphosphate