dUTP pyrophosphatase is an essential enzyme in Saccharomyces cerevisiae.

Gadsden, Michael H.; McIntosh, Evan M.; Game, John C.; Wilson, Paul J.; Haynes, Robert H.

doi:10.1002/j.1460-2075.1993.tb06127.x

Cited by 206 publications

(166 citation statements)

References 47 publications

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“…However, amelioration of futile BER cycling during TS inhibition by limiting UDG activity has its own limitation, namely that the presence of genomic uracil itself is dramatically problematic. The inviability of dUTPase deficient strains of E. coli and S. cerevisiae even in the absence of UDG activity suggest that excessive genomic uracil cannot be tolerated [31,32]. Studies that manipulated dUTPase levels during chemotherapy-induced thymidylate deprivation indicate that increased dUTPase activity can only delay death [18][19][20][21].…”

Section: Discussionmentioning

confidence: 99%

Uracil incorporation into genomic DNA does not predict toxicity caused by chemotherapeutic inhibition of thymidylate synthase

Luo¹,

Walla²,

Wyatt³

2008

DNA Repair

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Thymidylate synthase (TS) is an important target of several chemotherapeutic agents, including 5-FU and raltitrexed (Tomudex). During TS inhibition, TTP levels decrease with a subsequent increase in dUTP. Uracil incorporated into the genome is removed by base excision repair (BER). Thus, BER initiated by uracil DNA glycosylase (UDG) activity has been hypothesized to influence the toxicity induced by TS inhibitors. In this study we created a human cell line expressing the Ugi protein inhibitor of UNG family of UDGs, which reduces cellular UDG activity by at least 45-fold. Genomic uracil incorporation was directly measured by mass spectrometry following treatment with TS inhibitors. Genomic uracil levels were increased over 4-fold following TS inhibition in the Ugiexpressing cells, but did not detectably increase in UNG proficient cells. Despite the difference in genomic uracil levels, there was no difference in toxicity between the UNG proficient and UNGinhibited cells to folate or nucleotide-based inhibitors of TS. Cell cycle analysis showed that UNG proficient and UNG-inhibited cells arrested in early S-phase and resumed replication progression during recovery from RTX treatment almost identically. The induction of γ-H2AX was measured following TS inhibition as a measure of whether uracil excision promoted DNA double strand break formation during S-phase arrest. Although γ-H2AX was detectable following TS inhibition, there was no difference between UNG proficient and UNG-inhibited cells. We therefore conclude that uracil excision initiated by UNG does not adequately explain the toxicity caused by TS inhibition in this model.

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Section: Discussionmentioning

confidence: 99%

Uracil incorporation into genomic DNA does not predict toxicity caused by chemotherapeutic inhibition of thymidylate synthase

Luo¹,

Walla²,

Wyatt³

2008

DNA Repair

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“…The enzyme influences DNA synthesis in a crucial way and the existence of dUTPase has been shown to be important for the cell cycle. Attempts to completely inactivate the dUTPase gene in Escherichia coli (E. coli) and Saccharomyces cerevisiae have been unsuccessful (EIHajj et al, 1988;Gadsden et al, 1993). dUTPase keeps the intracellular concentration of dUTP at a low level thus suppressing the incorporation of uracil into DNA (Tye et al, 1977).…”

Section: Introductionmentioning

confidence: 99%

The Refined Structure of dUTPase from Escherichia coli

Dauter¹,

Wilson²,

Larsson³

et al. 1998

Acta Cryst D

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Deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase, E.C. 3.6.1.23) catalyzes the hydrolysis of dUTP to dUMP and pyrophosphate and is involved in nucleotide metabolism and DNA synthesis. A crystal of the recombinant E. coli enzyme, precipitated from polyethylene glycol mixtures in the presence of succinate at pH 4.2, was used to collect synchrotron diffraction data to 1.9 A resolution, in space group R3, a = b = 86.62, c = 62.23 A. Mercury and platinum derivative data were collected at wavelengths to optimize the anomalous contribution. The resulting 2.2 A MIRAS phases differed from the final set by 40 c on average and produced an excellent map which was easy to interpret. The model contains 132 water molecules and refined to an R value of 13.7%. 136 residues have clear electron density out of 152 expected from the gene sequence. The 16 C-terminal residues are presumably disordered in the crystal lattice. The monomer is a 'jelly-roll' type, containing mostly/3-sheet and only one short helix. The molecule is a tight trimer. A long C-terminal arm extends from one subunit and encompasses the next one within the trimer contributing to its fl-sheet. Conserved sequence motifs common among dUTPases, previously suggested to compose the active site and confirmed in a recent study of the dUDP complex, are located at subunit-subunit interfaces along the threefold axis, in parts of the fl-sheet and in loop regions. A similar molecular architecture has recently been found in two other trimeric dUTPases.

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“…Lack of the enzyme leads to massive uracil incorporation into DNA, repair of which via base excision, an efficient pathway to remove uracil, progresses to a hyperactive futile cycle resulting in DNA double-strand breaks, chromosome fragmentation, and cell death (4,5). dUTPase is therefore reported to be a preventive DNA repair factor (6), essential in both prokaryotes (7) and eukaryotes (8), and a key determinant of viral host cell range and infectivity of Herpes simplex virus (9), as well as several retroviruses (10 -12).…”

mentioning

confidence: 99%

Altered Active Site Flexibility and a Structural Metal-binding Site in Eukaryotic dUTPase

Kovari

Barábas

Takács

et al. 2004

Journal of Biological Chemistry

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dUTPase is responsible for preventive DNA repair via exclusion of uracil. Developmental regulation of the Drosophila enzyme is suggested to be involved in thymine-less apoptosis. Here we show that in addition to conserved dUTPase sequence motifs, the gene of Drosophila enzyme codes for a unique Ala-Pro-rich segment. Kinetic and structural analyses of the recombinant protein and a truncation mutant show that the Ala-Pro segment is flexible and has no regulatory role in vitro. The homotrimer enzyme unfolds reversibly as a trimeric entity with a melting temperature of 54°C, 23°C lower than Escherichia coli dUTPase. In contrast to the bacterial enzyme, Mg 2؉ binding modulates conformation of fly dUTPase, as identified by spectroscopy and by increment in melting temperature. A single well folded, but inactive, homotrimeric core domain is generated through three distinct steps of limited trypsinolysis. In fly, but not in bacterial dUTPase, binding of the product dUMP induces protection against proteolysis at the tryptic site reflecting formation of the catalytically competent closed conformer. Crystallographic analysis argues for the presence of a stable monomer of Drosophila dUTPase in crystal phase. The significant differences between prototypes of eukaryotic and prokaryotic dUTPases with respect to conformational flexibility of the active site, substrate specificity, metal ion binding, and oligomerization in the crystal phase are consistent with alteration of the catalytic mechanism and hydropathy of subunit interfaces.

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dUTP pyrophosphatase is an essential enzyme in Saccharomyces cerevisiae.

Cited by 206 publications

References 47 publications

Uracil incorporation into genomic DNA does not predict toxicity caused by chemotherapeutic inhibition of thymidylate synthase

Uracil incorporation into genomic DNA does not predict toxicity caused by chemotherapeutic inhibition of thymidylate synthase

The Refined Structure of dUTPase from Escherichia coli

Altered Active Site Flexibility and a Structural Metal-binding Site in Eukaryotic dUTPase

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