Depletion of dimeric all-α dUTPase induces DNA strand breaks and impairs cell cycle progression in Trypanosoma brucei

Abstract

The enzyme deoxyuridine 5′-triphosphate nucleotidohydrolase (dUTPase) is responsible for the control of intracellular levels of dUTP thus controlling the incorporation of uracil into DNA during replication. Trypanosomes and certain eubacteria contain a dimeric dUTP-dUDPase belonging to the recently described superfamily of all-α NTP pyrophosphatases which bears no resemblance with typical eukaryotic trimeric dUTPases and presents unique properties regarding substrate specificity and product inhibition. While the biological trimeric enzymes have been studied in detail and the human enzyme has been proposed as a promising novel target for anticancer chemotherapeutic strategies, little is known regarding the biological function of dimeric proteins. Here, we show that in Trypanosoma brucei, the dimeric dUTPase is a nuclear enzyme and that down-regulation of activity by RNAi greatly reduces cell proliferation and increases the intracellular levels of dUTP. Defects in growth could be partially reverted by the addition of exogenous thymidine. dUTPase-depleted cells presented hypersensitivity to methotrexate, a drug that increases the intracellular pools of dUTP, and enhanced uracil-DNA glycosylase activity, the first step in base excision repair. The knockdown of activity produces numerous DNA strand breaks and defects in both S and G2/M progression. Multiple parasites with a single enlarged nucleus were visualized together with an enhanced population of anucleated cells. We conclude that dimeric dUTPases are strongly involved in the control of dUTP incorporation and that adequate levels of enzyme are indispensable for efficient cell cycle progression and DNA replication.