An enzymatic method for distinguishing deoxyuridine and deoxythymidine nucleotide pools and its application for determining ribonucleotide reductase activity.
A method is described for distinguishing deoxyuridine and deoxythymidine di- and triphosphate pools. The method utilizes a DNA polymerase assay for triphosphate determination and a coupled assay in which the disphosphate is converted to its corresponding triphosphate by nucleoside-diphosphate kinase and the triphosphate is measured by the DNA polymerase assay. By including deoxyruidine-triphosphate nucleotidohydrolase in the reaction mixture, dUTP is removed as a substrate for the polymerase. By determining differences in labelled acid-insoluble product formed in the reaction it is possible to determine dUTP, dUDP, dTDP and dTTP pools. Ribonucleotide reductase activity was determined by converting either CDP or ADP to its corresponding deoxyribonucleoside disphosphate and then using the diphosphate assay described for deoxyribonucleoside pools.
D002277:Carcinoma; D002460:Cell Line; D004259:DNA-Directed DNA Polymerase; D003856:Deoxyuracil Nucleotides / Q000032:analysis*; D006801:Humans; D007700:Kinetics; D008722:Methods; D009062:Mouth Neoplasms; D012264:Ribonucleotide Reductases / Q000032:analysis*; D013942:Thymine Nucleotides / Q000032:analysis*
D003856:Deoxyuracil Nucleotides; D013942:Thymine Nucleotides; D012264:Ribonucleotide Reductases; D004259:DNA-Directed DNA Polymerase