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.

Introduction

Trypanosomes are protozoan parasites that cause major diseases in humans and other animals. Trypanosoma brucei is the etiologic agent of African Trypanosomiasis and although aspects of its biology have been studied in detail, many features of the mechanisms underlying DNA metabolism and repair remain to be established. The knowledge of mechanisms that maintain genetic integrity and generate genetic diversity is critical for understanding how trypanosomes survive within their hosts (Barry and McCulloch, 2001).

The enzyme deoxyuridine triphosphate nucleotidohydrolase (dUTPase) (EC 3.6.1.23) catalyzes the hydrolysis of dUTP to dUMP and PPi and has a dual role in pyrimidine nucleotide metabolism. Firstly, it provides the substrate for the thymidylate synthase allowing the formation of dTTP. Secondly, the enzyme is responsible for keeping dUTP intracellular levels low, avoiding its misincorporation into nascent DNA. The ability to specifically remove the non-canonical nucleotide, dUTP, from the dNTP pool suggests the importance of this enzyme in the preservation of genetic integrity. Clearly, inhibition of dUTPase would be damaging to the cell and has been recognized as a potential means of slowing viral replication and cancer growth (McIntosh and Haynes, 1997, Grasser et al., 2001, Ladner, 2001). Trimeric dUTPases such as the human and Escherichia coli enzymes, present a unique active site architecture, where all three monomers contribute to each of the three catalytic sites (Persson et al., 2001). Trypanosomes are to date the only eukaryote that exhibits a dimeric form of dUTPase that has an entirely different 3D structure (Harkiolaki et al., 2004, Moroz et al., 2004) and is an analogue, not a homologue, of trimeric dUTPases such as the human enzyme. Homologues of the dimeric enzyme have been identified in the genomes of several Gram-positive bacteria and their phages (Moroz et al., 2004) showing that this class of enzymes is not restricted to parasites of the Trypanosomatidae family. Furthermore, structure-guided analysis of the new dimeric dUTPase family revealed its sequence relationship to the phage T4 dCTPase, phosphoribosyl-ATP pyrophosphatase HisE, MazG, and several uncharacterized protein families, including the human protein XTP3TPA (RS21-C6). This observation led to the unification of these enzymes in a superfamily of all-α NTP pyrophosphatases and its classification as one of the structural superfamilies belonging to house-cleaning NTP pyrophosphatases (Moroz et al., 2005, Galperin et al., 2006). Dimeric dUTPases differ profoundly from the classical trimeric dUTPase in a number of features, including the ability to use both dUTP and dUDP as substrates, with dUMP being the product in each case, and the existence of product inhibition (Hidalgo-Zarco et al., 2001).

Uracil incorporation is enhanced under conditions of reduced dTTP biosynthesis (caused by folate deficiency or by thymidylate synthetase inhibition), is detrimental to prokaryotic and eukaryotic cells (Ahmad et al., 1998, Fenech, 2001) and produces chromosomal instability (Blount et al., 1997, Duthie et al., 2004). The existence of controlled mechanisms of uracil-induced genetic instability and cell death are yet to be fully explored at both the molecular and chromosomal levels. It is known that uracil-mediated base excision DNA occurs as a strategic cellular defense mechanism to maintain the genetic stability of the genome in different cell types (Mosbaugh and Bennett, 1994). Conversely, the presence of uracil is important for somatic hypermutation and class switch recombination in adaptive immunity (Di Noia and Neuberger, 2007). In addition, this base is generated in retroviral DNA by the host as part of a defense mechanism (Bishop et al., 2004, Mangeat et al., 2003). Base excision repair is initiated when uracil-DNA glycosylase recognizes a uracil residue in DNA and catalyzes the cleavage of the N-glycosylic bond that links the uracil base to the deoxyribose phosphate DNA backbone (Lindahl et al., 1977). This hydrolytic reaction results in the release of free uracil and creates an abasic site in the DNA (Lindahl et al., 1977). Incision by a class II AP endonuclease, cleaves the phosphodiester bond on the 5′-side of the AP site to generate a terminal 3′-hydroxyl-containing nucleotide and a deoxyribose 5′-phosphate residue (Lindahl, 2001). A single uracil-DNA glycosylase while two class II AP endonucleases are present in the genome of T. brucei, Trypanosoma cruzi and Leishmania major (www.GeneDB.org). The apurinic/apyrimidinic endonuclease of L. major is a key element in mediating repair of apurinic/apyrimidinic sites and 3′-blocked termini (Vidal et al., 2007) while the cleavage of uracil by uracil-DNA glycosylase has been shown in T. cruzi (Pena-Diaz et al., 2004).

The trimeric dUTPase is essential for cell viability in E. coli and has a critical role in viability and the maintenance of genetic stability in Saccharomyces cerevisiae (Gadsden et al., 1993, Guillet et al., 2006). Indeed these studies show that dUTP, a nucleotide produced by normal cellular metabolism, is an important source of endogenous DNA damage. Despite their unique character, no study has been performed to date to determine if dimeric proteins perform a similar role in controlling intracellular levels of dUTP and adequate cell proliferation.

In the present study, we have used the RNAi technique to knockdown dUTPase expression in T. brucei. We show that dUTPase-depleted procyclic cells show delayed growth, multiple cell cycle abnormalities including defects in cytokinesis, DNA strand breaks and increased uracil-DNA glycosylase activity. Moreover, we demonstrate that dUTPase localizes to the nucleus suggesting that it is involved in controlling dUTP incorporation during replication. The data obtained indicates an important role of this enzyme in the control of intracellular dUTP levels and in the progression of cell division and contribute to understanding how trypanosomes control the presence of uracil in DNA.