Human dUTP pyrophosphatase: cDNA sequence and potential biological importance of the enzyme.

McIntosh, Evan M.; Ager, D.D.; Gadsden, Michael H.; Haynes, Robert H.

doi:10.1073/pnas.89.17.8020

Cited by 65 publications

(50 citation statements)

References 31 publications

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“…1 An additional enzymatic activity, deamination of dCTP (deoxycytidine triphosphate) to dUTP, may also be carried out by bifunctional dUTPases in Archaea [2][3][4][5] and also in Mycobacterium tuberculosis (M. tuberculosis), which also has a monofunctional dUTPase. 6 The dUTPases comprise two structurally different classes: 7 first, the β sheet−type dUTPases, possessed by most organisms, employ a one-metal ion catalytic mechanism, [8][9] and usually form a homotrimer 10 or a monomer, 11 while the second class, the α helical-type dUTPases, possessed by kinetoplastids, 12 form dimers and employ a two-metal ion catalytic mechanism.…”

Section: Introductionmentioning

confidence: 99%

Mutations Decouple Proton Transfer from Phosphate Cleavage in the dUTPase Catalytic Reaction

et al. 2015

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Most enzymes present a catalytic mechanism where one or more proton transfer events occur coupled tightly together with the enzymatic chemical reaction. We show here that inactivating mutations decouple this proton transfer step from the phosphate cleavage reaction in dUTPase. Homotrimeric dUTPase enzymes catalyze the hydrolysis of dUTP to dUMP and pyrophosphate, using largely similar structural and functional groups as most AAA+ enzymes. dUTPases typically use a single Mg 2+ ion as a cofactor in the active site that is formed by direct protein-protein contacts including all three protomers. Here we focus on the C-terminal arm structural motif, which has sequence and functional similarities to P-loop motifs, and is required for catalysis. In this work, we have studied the functional roles of the C-terminal arm in ligand binding and catalysis by using QM/MM (quantum mechanics/molecular mechanics) calculations in conjunction with site-directed mutagenesis experiments. We also present a new method to assess the metal ion coordination symmetry during the catalytic reaction. Using this new implementation, we identified that the coordination symmetry follows a consistent pattern in the three systems studied, reaching the most symmetrical state near the transition states. We found that the phosphate cleavage proceeds with a concerted bimolecular (A N D N ) mechanism with a loose dissociative transition state, and that it is coupled with a proton transfer step involving a unanimously conserved Asp residue. We show that the main mechanistic effect of the lack of the C-terminal arm is to decouple the phosphate cleavage from the subsequent proton transfer step, resulting in a highbarrier altered reaction pathway.

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Section: Introductionmentioning

confidence: 99%

Mutations Decouple Proton Transfer from Phosphate Cleavage in the dUTPase Catalytic Reaction

et al. 2015

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“…Although the resulting U/A pairs are not inherently mutagenic, stringent control on uracil incorporation is required because high levels of uracil in DNA can lead to genomic instability (4-7). The primary barrier to dUTP incorporation is the enzyme dUTPase, which hydrolyzes dUTP to dUMP and pyrophosphate (8). Subsequent reductive methylation of dUMP by thymidylate synthase (TS) generates dTMP, which is then phosphorylated to produce dTTP.…”

mentioning

confidence: 99%

Uracil DNA glycosylase initiates degradation of HIV-1 cDNA containing misincorporated dUTP and prevents viral integration

Weil

Ghosh²,

Zhou

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

119

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Significance Misincorporation of dUTP into DNA is detrimental to eukaryotes, prokaryotes, and viruses. This study reveals the fate of uracilated HIV-1 DNA in human cells. Early stages of the viral life cycle are unaffected, but integration of uracilated viral DNA into the host genome is prevented. This effect is wholly dependent on the presence of UNG2, a nuclear enzyme that excises uracil from DNA. This study establishes that UNG2 is a restriction factor for uracilated HIV-1 DNA and explains why this pathway is not fully engaged in CD4+ T cells and macrophages.

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“…These enzymes catalyze the hydrolysis of the nucleotide triphosphate to its monophosphate and pyrophosphate, simultaneously removing aberrant dNTPs (dUTP or 8-oxodGTP) during DNA synthesis. The importance of the function of dUTPase and MutT in prokaryotic and eukaryotic systems has been firmly established (McIntosh et al, 1992;Larsson et al, 1996;Bridges, 1997;Fowler and Schaaper, 1997). It is notable that the EcO197 protein also releases pyrophosphate from dITP and XTP in the same way as dUTPase and MutT.…”

Section: Eco197mentioning

confidence: 98%

“…This nucleotide-cleansing function has been seen for dUTPase (McIntosh et al, 1992;Larsson et al, 1996) and MutT (Bridges, 1997;Fowler and Schaaper, 1997), removing dUTP and 8-oxo-dGTP from the nucleotide pool, respectively. These enzymes catalyze the hydrolysis of the nucleotide triphosphate to its monophosphate and pyrophosphate, simultaneously removing aberrant dNTPs (dUTP or 8-oxodGTP) during DNA synthesis.…”

Section: Eco197mentioning

confidence: 99%

“…Typical enzymes that can hydrolyze (d)NTP to (d)NMP and PPi are MutT (Maki and Sekiguchi, 1992;Bridges, 1997) and dUTPase (McIntosh et al, 1992;Larsson et al, 1996), eliminating 8-oxo-dGTP and dUTP from the nucleotide precursor pools, respectively. The importance of these DNA repair enzymes in bacteria and eukaryotes has been well established (Maki and Sekiguchi, 1992;McIntosh et al, 1992;Larsson et al, 1996;Bridges, 1997). However, repair proteins that deal with the damaged nucleoside triphosphates that are produced by oxidative deamination, such as dITP or XTP, have been unknown until now.…”

Section: Introductionmentioning

confidence: 99%

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Identificaiton of the dITP- and XTP-Hydrolyzing Protein from Escherichia coli

Chung¹,

Park²,

Lee³

et al. 2002

BMB Reports

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A hypothetical 21.0 kDa protein (ORF O197) fromEscherichia coli K-12 was cloned, purified, and characterized. The protein sequence of ORF O197 (termed EcO197) shares a 33.5% identity with that of a novel NTPase from Methanococcus jannaschii. The EcO197 protein was purified using Ni-NTA affinity chromatography, protease digestion, and gel filtration column. It hydrolyzed nucleoside triphosphates with an O6 atom-containing purine base to nucleoside monophosphate and pyrophosphate. The EcO197 protein had a strong preference for deoxyinosine triphosphate (dITP) and xanthosine triphosphate (XTP), while it had little activity in the standard nucleoside triphosphates (dATP, dCTP, dGTP, and dTTP). These aberrant nucleotides can be produced by oxidative deamination from purine nucleotides in cells; they are potentially mutagenic. The mutation protection mechanisms are caused by the incorporation into DNA of unwelcome nucleotides that are formed spontaneously. The EcO197 protein may function to eliminate specifically damaged purine nucleotide that contains the 6-keto group. This protein appears to be the first eubacterial dITP-and XTPhydrolyzing enzyme that has been identified.

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Human dUTP pyrophosphatase: cDNA sequence and potential biological importance of the enzyme.

Abstract: Two functional human dUTP pyrophosphatse (dUTPase; EC 3.6.

Cited by 65 publications

References 31 publications

Mutations Decouple Proton Transfer from Phosphate Cleavage in the dUTPase Catalytic Reaction

Mutations Decouple Proton Transfer from Phosphate Cleavage in the dUTPase Catalytic Reaction

Uracil DNA glycosylase initiates degradation of HIV-1 cDNA containing misincorporated dUTP and prevents viral integration

Identificaiton of the dITP- and XTP-Hydrolyzing Protein from Escherichia coli

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