A SIMPLE AND SENSITIVE RIBONUCLEOTIDE REDUCTASE ASSAY

Ribonucleotide reductase (RR) is a key regulatory enzyme in the DNA sy nthesis 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 pre sent a novel RR assay in which detection of the deoxyribonucleotides p roduced by RR occurs via coupling to the DNA polymerase reaction, and is enhanced by using RNase to degrade endogenous RNA. Cell extracts fr om various cell lines were treated with RNase and then reacted with AT P and radioactive ribonucleotide diphosphate as the substrate. Incorpo ration of the radioactive substrate [C-14]CDP into DNA was linear over 30 min and was linear with the amount of extract, which provided RR a ctivity. The reaction was inhibited by hydroxyurea and required Mg2+ a nd 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 sens itive and less expensive. In addition, assay of the RR activity for mu ltiple samples can easily be performed simultaneously. It is superior to other RR assays in all aspects.