A simple and sensitive ribonucleotide reductase assay.
Ribonucleotide reductase (RR) is a key regulatory enzyme in the DNA synthesis pathway and is the target of the cancer chemotherapeutic agent hydroxyurea. The study of RR is significantly hindered by the tedious and labor-intensive nature of enzymatic assay. In this report, we present a novel RR assay in which detection of the deoxyribonucleotides produced by RR occurs via coupling to the DNA polymerase reaction, and is enhanced by using RNase to degrade endogenous RNA. Cell extracts from various cell lines were treated with RNase and then reacted with ATP and radioactive ribonucleotide diphosphate as the substrate. Incorporation of the radioactive substrate [14C]CDP into DNA was linear over 30 min and was linear with the amount of extract, which provided RR activity. The reaction was inhibited by hydroxyurea and required Mg2+ and ATP, suggesting that the assay is specific to RR activity. While RR activities determined by our method and by a conventional method were comparable, this novel method proved to be simpler, faster, more sensitive and less expensive. In addition, assay of the RR activity for multiple samples can easily be performed simultaneously. It is superior to other RR assays in all aspects.
Jong
A Y
AY
Department of Pediatrics, Childrens Hospital of Los Angeles and the University of Southern California School of Medicine, 90027, USA. ajong%smtpgate@chlais.usc.edu
Yu
K
K
Zhou
B
B
Frgala
T
T
Reynolds
C P
CP
Yen
Y
Y
D002138:Calibration; D002250:Carbon Radioisotopes; D002460:Cell Line; D002999:Clone Cells; D003565:Cytidine Diphosphate / Q000378:metabolism; D004247:DNA / Q000096:biosynthesis; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D006801:Humans; D007624:KB Cells; D007700:Kinetics; D008297:Male; D009447:Neuroblastoma; D009959:Oropharyngeal Neoplasms; D011471:Prostatic Neoplasms; D011865:Radioisotope Dilution Technique; D012260:Ribonucleases / Q000378:metabolism; D012264:Ribonucleotide Reductases / Q000032:analysis* / Q000378:metabolism; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured
D002250:Carbon Radioisotopes; D003565:Cytidine Diphosphate; D004247:DNA; D012264:Ribonucleotide Reductases; D004259:DNA-Directed DNA Polymerase; D012260:Ribonucleases