Mycobacterium tuberculosis MutT1 (Rv2985) and ADPRase (Rv1700) proteins constitute a two-stage mechanism of 8-oxo-dGTP and 8-oxo-GTP detoxification and adenosine to cytidine mutation avoidance.
ABSTRACT: Approximately one third of the world population is infected with Mycobacterium tuberculosis, the causative agent of tuberculosis. A better understanding of the pathogen biology is crucial to develop new tools/strategies to tackle its spread and treatment. In the host macrophages, the pathogen is exposed to reactive oxygen species, known to damage dGTP and GTP to 8-oxo-dGTP and 8-oxo-GTP, respectively. Incorporation of the damaged nucleotides in nucleic acids is detrimental to organisms. MutT proteins, belonging to a class of Nudix hydrolases, hydrolyze 8-oxo-G nucleoside triphosphates/diphosphates to the corresponding nucleoside monophosphates and sanitize the nucleotide pool. Mycobacteria possess several MutT proteins. However, a functional homolog of Escherichia coli MutT has not been identified. Here, we characterized MtuMutT1 and Rv1700 proteins of M. tuberculosis. Unlike other MutT proteins, MtuMutT1 converts 8-oxo-dGTP to 8-oxo-dGDP, and 8-oxo-GTP to 8-oxo-GDP. Rv1700 then converts them to the corresponding nucleoside monophosphates. This observation suggests the presence of a two-stage mechanism of 8-oxo-dGTP/8-oxo-GTP detoxification in mycobacteria. MtuMutT1 converts 8-oxo-dGTP to 8-oxo-dGDP with a Km of ?50 ?M and Vmax of ?0.9 pmol/min per ng of protein, and Rv1700 converts 8-oxo-dGDP to 8-oxo-dGMP with a Km of ?9.5 ?M and Vmax of ?0.04 pmol/min per ng of protein. Together, MtuMutT1 and Rv1700 offer maximal rescue to E. coli for its MutT deficiency by decreasing A to C mutations (a hallmark of MutT deficiency). We suggest that the concerted action of MtuMutT1 and Rv1700 plays a crucial role in survival of bacteria against oxidative stress.
Project description:Most of the proteins carrying the 23-residue MutT-related sequence are capable of hydrolyzing compounds with a general structure of nucleoside diphosphate linked to another moiety X and are called the Nudix hydrolases. Among the 22 human Nudix proteins (identified by the sequence signature), some remain uncharacterized as enzymes without a defined substrate. Here, we reveal that the NUDT18 protein, whose substrate was unknown, can degrade 8-oxo-7,8-dihydroguanine (8-oxo-Gua)-containing nucleoside diphosphates to the monophosphates. Because this enzyme is closely related to MTH1 (NUDT1) and MTH2 (NUDT15), we propose that it should be named MTH3. Although these three human proteins resemble each other in their sequences, their substrate specificities differ considerably. MTH1 cleaves 8-oxo-dGTP but not 8-oxo-dGDP, whereas MTH2 can degrade both 8-oxo-dGTP and 8-oxo-dGDP, although the intrinsic enzyme activity of MTH2 is considerably lower than that of MTH1. On the other hand, MTH3 is specifically active against 8-oxo-dGDP and hardly cleaves 8-oxo-dGTP. Other types of oxidized nucleoside diphosphates, 2-hydroxy-dADP and 8-hydroxy-dADP, were also hydrolyzed by MTH3. Another notable feature of the MTH3 enzyme is its action toward the ribonucleotide counterpart. MTH3 can degrade 8-oxo-GDP as efficiently as 8-oxo-dGDP, which is in contrast to the finding that MTH1 and MTH2 show a limited activity against the ribonucleotide counterpart, 8-oxo-GTP. These three enzymes may function together to help maintain the high fidelity of DNA replication and transcription under oxidative stress.
Project description:The chemical integrity of the nucleotide pool and its homeostasis are crucial for genome stability. Nucleoside diphosphate kinase (NDK) is a crucial enzyme that carries out reversible conversions from nucleoside diphosphate (NDP) to nucleoside triphosphate (NTP) and deoxynucleoside diphosphate (dNDP) to deoxynucleoside triphosphate (dNTP). Guanosine nucleotides (GDP, GTP, dGDP, and dGTP) are highly susceptible to oxidative damage to 8-oxo-GDP (8-O-GDP), 8-O-dGTP, 8-O-GTP, and 8-O-dGTP. MutT proteins in cells hydrolyze 8-O-GTP to 8-O-GMP or 8-O-dGTP to 8-O-dGMP to avoid its incorporation in nucleic acids. In <i>Escherichia coli</i>, 8-O-dGTP is also known to be hydrolyzed by RibA (GTP cyclohydrolase II). In this study, we show that <i>E. coli</i> NDK catalyzes the conversion of 8-O-dGDP to 8-O-dGTP or vice versa. However, the rate of NDK-mediated phosphorylation of 8-O-dGDP to 8-O-dGTP is about thrice as efficient as the rate of dephosphorylation of 8-O-dGTP to 8-O-dGDP, suggesting an additive role of NDK in net production of 8-O-dGTP in cells. Consistent with this observation, the depletion of NDK (?<i>ndk</i>) in <i>E. coli</i> ?<i>mutT</i> or ?<i>mutT</i> ?<i>ribA</i> strains results in a decrease of A-to-C mutations. These observations suggest that NDK contributes to the physiological load of MutT in <i>E. coli</i> <b>IMPORTANCE</b> Nucleoside diphosphate kinase (NDK), a ubiquitous enzyme, is known for its critical role in homeostasis of cellular nucleotide pools. However, NDK has now emerged as a molecule with pleiotropic effects in DNA repair, protein phosphorylation, gene expression, tumor metastasis, development, and pathogen virulence and persistence inside the host. In this study, we reveal an unexpected role of NDK in genome instability because of its activity in converting 8-O-dGDP to 8-O-dGTP. This observation has important consequences in escalating A-to-C mutations in <i>Escherichia coli</i> The severity of NDK in enhancing these mutations may be higher in the organisms challenged with high oxidative stress, which promotes 8-O-dGDP/8-O-dGTP production.
Project description:Mycobacterium tuberculosis, the causative agent of tuberculosis, is at increased risk of accumulating damaged guanine nucleotides such as 8-oxo-dGTP and 8-oxo-GTP because of its residency in the oxidative environment of the host macrophages. By hydrolyzing the oxidized guanine nucleotides before their incorporation into nucleic acids, MutT proteins play a critical role in allowing organisms to avoid their deleterious effects. Mycobacteria possess several MutT proteins. Here, we purified recombinant M. tuberculosis MutT2 (MtuMutT2) and M. smegmatis MutT2 (MsmMutT2) proteins from M. tuberculosis (a slow grower) and M. smegmatis (fast growing model mycobacteria), respectively, for their biochemical characterization. Distinct from the Escherichia coli MutT, which hydrolyzes 8-oxo-dGTP and 8-oxo-GTP, the mycobacterial proteins hydrolyze not only 8-oxo-dGTP and 8-oxo-GTP but also dCTP and 5-methyl-dCTP. Determination of kinetic parameters (Km and Vmax) revealed that while MtuMutT2 hydrolyzes dCTP nearly four times better than it does 8-oxo-dGTP, MsmMutT2 hydrolyzes them nearly equally. Also, MsmMutT2 is about 14 times more efficient than MtuMutT2 in its catalytic activity of hydrolyzing 8-oxo-dGTP. Consistent with these observations, MsmMutT2 but not MtuMutT2 rescues E. coli for MutT deficiency by decreasing both the mutation frequency and A-to-C mutations (a hallmark of MutT deficiency). We discuss these findings in the context of the physiological significance of MutT proteins.
Project description:Human MutT homologue 1 (hMTH1) hydrolyzes a variety of oxidized purine nucleoside triphosphates, including 8-oxo-dGTP, 2-oxo-dATP, 2-oxo-ATP and 8-oxo-dATP, to their corresponding nucleoside monophosphates, while Escherichia coli MutT possesses prominent substrate specificity for 8-oxoguanine nucleotides. Three types of crystals were obtained corresponding to the following complexes: selenomethionine-labelled hMTH1 with 8-oxo-dGMP (SeMet hMTH1-8-oxo-dGMP), hMTH1 with 8-oxo-dGMP (hMTH1-8-oxo-dGMP) and hMTH1 with 2-oxo-dATP (hMTH1-2-oxo-dATP). Crystals of the SeMet hMTH1-8-oxo-dGMP complex belong to space group P4(1)2(1)2, with unit-cell parameters a = b = 45.8, c = 153.6 A, and diffracted to 2.90 A. Crystals of hMTH1-8-oxo-dGMP and hMTH1-2-oxo-dATP belong to space groups P2(1) and P2(1)2(1)2(1), with unit-cell parameters a = 34.0, b = 59.0, c = 65.9 A, beta = 90.7 degrees and a = 59.2, b = 67.3, c = 80.0 A, respectively. Their diffraction data were collected at resolutions of 1.95 and 2.22 A, respectively.
Project description:Living in an oxygen-rich environment is dangerous for a cell. Reactive oxygen species can damage DNA, RNA, protein and lipids. The MutT protein in Escherichia coli removes 8-oxo-deoxyguanosine triphosphate (8-oxo-dGTP) and 8-oxo-guanosine triphosphate (8-oxo-GTP) from the nucleotide pools precluding incorporation into DNA and RNA. While 8-oxo-dGTP incorporation into DNA is mutagenic, it is not clear if 8-oxo-GTP incorporation into RNA can have phenotypic consequences for the cell. We use a bistable epigenetic switch sensitive to transcription errors in the Escherichia coli lacI transcript to monitor transient RNA errors. We do not observe any increase in epigenetic switching in mutT cells. We revisit the original observation of partial phenotypic suppression of a lacZamber allele in a mutT background that was attributed to RNA errors. We find that Lac+ revertants can completely account for the increase in ?-galactosidase levels in mutT lacZamber cultures, without invoking participation of transient transcription errors. Moreover, we observe a fluctuation type of distribution of ?-galactosidase appearance in a growing culture, consistent with Lac+ DNA revertant events. We conclude that the absence of MutT produces a DNA mutator but does not equally create an RNA mutator.
Project description:8-Oxo-dGTP, an oxidised form of dGTP generated in the nucleotide pool, can be incorporated opposite adenine or cytosine in template DNA, which can in turn induce mutations. In this study, we identified a novel MutT homolog (NDX-2) of Caenorhabditis elegans that hydrolyzes 8-oxo-dGDP to 8-oxo-dGMP. In addition, we found that NDX-1, NDX-2 and NDX-4 proteins have 8-oxo-GTPase or 8-oxo-GDPase activity. The sensitivity of ndx-2 knockdown C. elegans worms to methyl viologen and menadione bisulphite was increased compared with that of control worms. This sensitivity was rescued by depletion of chk-2 and clk-2, suggesting that growth of the worms is regulated by the checkpoint pathway in response to the accumulation of oxidised nucleotides. Moreover, we found that the sensitivity to menadione bisulphite of ndx-1 and ndx-2-double knockdown worms was enhanced by elimination of XPA-1, a factor involved in nucleotide excision repair. The rescue effect by depletion of chk-2 and clk-2 was limited in the xpa-1 mutant, suggesting that the chk-2 and clk-2 checkpoint pathway is partially linked to the function of XPA-1.
Project description:We have studied the kinetics of guanine incorporation into DNA in mouse T-lymphoma (S-49) mutant cells [PNPase (purine-nucleoside phosphorylase)- and HGPRTase (hypoxanthine: guanine phosphoribosyltransferase)-deficient] that are incapable of converting dGuo (deoxyguanosine) to Gua (guanine) ribonucleotides. Of the two possible pathways for an exogenous guanine source to reach DNA, firstly: dGuo----dGMP----dGDP----dGTP and secondly: Gua----GMP----GDP----dGDP----dGTP only the second pathway was found to be functional in providing guanine for DNA replication, although deoxyguanosine readily produced toxic cellular dGTP levels via the first pathway. The functional guanine-nucleotide-precursor pools for DNA are rather small; further, the depletion of the small GMP pool, but not that of GDP, GTP and dGTP, correlated well with the inhibition of DNA synthesis by mycophenolic acid, an IMP dehydrogenase inhibitor. These results support the hypothesis that guanine-nucleotide incorporation into DNA is highly compartmentalized and that a small functional guanine-nucleotide pool, e.g., the GMP pool, may serve a crucial role in limiting the availability of DNA precursor substrate.
Project description:Reactive oxygen species induce oxidative damage in DNA precursors, i.e. dNTPs, leading to point mutations upon incorporation. Escherichia coli mutT strains, deficient in the activity hydrolysing 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP), display more than a 100-fold higher spontaneous mutation frequency over the wild-type strain. 8-oxo-dGTP induces A to C transversions when misincorporated opposite template A. Here, we report that DNA pol III incorporates 8-oxo-dGTP ??20 times more efficiently opposite template A compared with template C. Single, double or triple deletions of pol I, pol II, pol IV or pol V had modest effects on the mutT mutator phenotype. Only the deletion of all four polymerases led to a 70% reduction of the mutator phenotype. While pol III may account for nearly all 8-oxo-dGTP incorporation opposite template A, it only extends ??30% of them, the remaining 70% being extended by the combined action of pol I, pol II, pol IV or pol V. The unique property of pol III, a C-family DNA polymerase present only in eubacteria, to preferentially incorporate 8-oxo-dGTP opposite template A during replication might explain the high spontaneous mutation frequency in E.?coli mutT compared with the mammalian counterparts lacking the 8-oxo-dGTP hydrolysing activities.
Project description:Oxidative stress promotes genomic instability and human diseases. A common oxidized nucleoside is 8-oxo-7,8-dihydro-2'-deoxyguanosine, which is found both in DNA (8-oxo-G) and as a free nucleotide (8-oxo-dGTP). Nucleotide pools are especially vulnerable to oxidative damage. Therefore cells encode an enzyme (MutT/MTH1) that removes free oxidized nucleotides. This cleansing function is required for cancer cell survival and to modulate Escherichia coli antibiotic sensitivity in a DNA polymerase (pol)-dependent manner. How polymerases discriminate between damaged and non-damaged nucleotides is not well understood. This analysis is essential given the role of oxidized nucleotides in mutagenesis, cancer therapeutics, and bacterial antibiotics. Even with cellular sanitizing activities, nucleotide pools contain enough 8-oxo-dGTP to promote mutagenesis. This arises from the dual coding potential where 8-oxo-dGTP(anti) base pairs with cytosine and 8-oxo-dGTP(syn) uses its Hoogsteen edge to base pair with adenine. Here we use time-lapse crystallography to follow 8-oxo-dGTP insertion opposite adenine or cytosine with human pol ?, to reveal that insertion is accommodated in either the syn- or anti-conformation, respectively. For 8-oxo-dGTP(anti) insertion, a novel divalent metal relieves repulsive interactions between the adducted guanine base and the triphosphate of the oxidized nucleotide. With either templating base, hydrogen-bonding interactions between the bases are lost as the enzyme reopens after catalysis, leading to a cytotoxic nicked DNA repair intermediate. Combining structural snapshots with kinetic and computational analysis reveals how 8-oxo-dGTP uses charge modulation during insertion that can lead to a blocked DNA repair intermediate.
Project description:Two alternative pathways for the synthesis of dGTP and its incorporation into DNA were studied: guanine (Gua)----GMP----GDP----dGDP----dGTP----DNA and dG----dGMP----dGDP----dGTP----DNA. To determine the contribution of each pathway to DNA synthesis independently of each other, [14C]Gua and [3H]dG tracer experiments were performed in a double-mutant S-49 mouse T-lymphoma cell line, dGuo-L, with purine nucleoside phosphorylase (EC 126.96.36.199)-deficiency and dGTP-feedback-resistant ribonucleotide reductase (RR, EC 188.8.131.52). In this cell line, dGTP pools can be selectively elevated by exogenous dG without affect RR and DNA synthesis. Although [3H]dG, but not [14C]Gua (up to 200 microM), readily expanded the cellular dGTP pool in a dose-dependent fashion in asynchronous cells, only a small fraction of the Gua flux into DNA was derived from [3H]dG, with the major fraction coming from [14C]Gua. H.p.l.c. analysis of G1- and partially enriched S-phase cells revealed that [3H]dGTP only accumulates in G1- but not in S-phase cells because of a rapid turnover of the dGTP pool during DNA synthesis. These results fail to provide evidence for cellular dGTP compartmentation and suggest that the pathway dG----dGMP----dGDP----dGTP alone has insufficient capacity to maintain DNA synthesis.