DUTP PYROPHOSPHATASE IS AN ESSENTIAL ENZYME IN SACCHAROMYCES-CEREVISIAE

dUTP pyrophosphatase (dUTPase; EC 3.6.1.23) catalyses the hydrolysis o f dUTP to dUMP and PP(i) and thereby prevents the incorporation of ura cil into DNA during replication. Although it is widely believed that d UTPase is essential for cell viability because of this role, direct ev idence supporting this assumption has not been presented for any eukar yotic system. We have analysed the role of dUTPase (DUT1) in the life cycle of yeast. Using gene disruption and tetrad analysis, we find tha t DUT1 is necessary for the viability of S.cerevisiae; however, under certain conditions dut1 null mutants survive if supplied with exogenou s thymidylate (dTMP). Analyses with isogenic uracil-DNA-glycosylase (U NG1) deficient or proficient strains indicate that in the absence of d UTPase, cell death results from the incorporation of uracil into DNA a nd the attempted repair of this damage by UNG1-mediated excision repai r. However, in dut1 ung1 double mutants, starvation for dTMP causes di viding cells to arrest and die in all phases of the cell cycle. This l atter effect suggests that the extensive stable substitution of uracil for thymine in DNA leads to a general failure in macromolecular synth esis. These results are in general agreement with previous models in t hymine-less death that implicate dUTP metabolism. They also suggest an alternative approach for chemotherapeutic drug design.