An isotopic assay of dUTPase activity based on coupling with thymidylate synthase

A new rapid, sensitive and convenient procedure is presented allowing deter mination of dUTPase activity. With [5-H-3]dUTP used as the substrate, dUTPa se, converts it to the corresponding monophosphate and is coupled with thym idylate synthase-catalyzed reaction, resulting in tritium release from [5-H -3]dUMP. Following charcoal absorption of the labeled nuleotides,radioactiv ity of tritiated water is determined. The new assay was tested to show comp arable results with a previously described assay, based on measuring dUTPas e-catalyzed [5-H-3]dUMP production.