Generation of reactive oxygen species and reactive nitrogen species in phagocytes is an important innate immune response mechanism to eliminate microbial pathogens. It is known that deoxynucleotides (dNTPs), the precursor nucleotides to DNA synthesis, are one group of the significant targets for these oxidants and incorporation of oxidized dNTPs into genomic DNA may cause mutations and even cell death. Here we show that the mycobacterial dNTP pyrophosphohydrolase MazG safeguards the bacilli genome by degrading 5-OH-dCTP, thereby, preventing it from incorporation into DNA. Deletion of the (d)NTP pyrophosphohydrolase-encoding mazG in mycobacteria leads to a mutator phenotype both under oxidative stress and in the stationary phase of growth, resulting in increased CG to TA mutations. Biochemical analyses demonstrate that mycobacterial MazG can efficiently hydrolyze 5-OH-dCTP, an oxidized nucleotide that induces CG to TA mutation upon incorporation by polymerase. Moreover, chemical genetic analyses show that direct incorporation of 5-OH-dCTP into mazG-null mutant strain of Mycobacterium smegmatis (Msm) leads to a dose-dependent mutagenesis phenotype, indicating that 5-OH-dCTP is a natural substrate of mycobacterial MazG. Furthermore, deletion of mazG in Mycobacterium tuberculosis (Mtb) leads to reduced survival in activated macrophages and in the spleen of infected mice. This study not only characterizes the mycobacterial MazG as a novel pyrimidine-specific housecleaning enzyme that prevents CG to TA mutation by degrading 5-OH-dCTP but also reveals a genome-safeguarding mechanism for survival of Mtb in vivo.
Vyšlo v časopise: Mycobacterial MazG Safeguards Genetic Stability Housecleaning of 5-OH-dCTP. PLoS Pathog 9(12): e32767. doi:10.1371/journal.ppat.1003814 Kategorie: Research Article prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.ppat.1003814
1. WagnerJR, HuCC, AmesBN (1992) Endogenous oxidative damage of deoxycytidine in DNA. Proc Natl Acad Sci U S A 89 : 3380–3384.
2. SekiguchiM, TsuzukiT (2002) Oxidative nucleotide damage: consequences and prevention. Oncogene 21 : 8895–8904.
3. HaghdoostS, SjolanderL, CzeneS, Harms-RingdahlM (2006) The nucleotide pool is a significant target for oxidative stress. Free Radic Biol Med 41 : 620–626.
4. NathanC, ShilohMU (2000) Reactive oxygen and nitrogen intermediates in the relationship between mammalian hosts and microbial pathogens. Proc Natl Acad Sci U S A 97 : 8841–8848.
5. FangFC (2004) Antimicrobial reactive oxygen and nitrogen species: concepts and controversies. Nat Rev Microbiol 2 : 820–832.
6. RaiP (2010) Oxidation in the nucleotide pool, the DNA damage response and cellular senescence: Defective bricks build a defective house. Mutat Res 703 : 71–81.
7. VenturaI, RussoMT, De LucaG, BignamiM (2010) Oxidized purine nucleotides, genome instability and neurodegeneration. Mutat Res 703 : 59–65.
8. MakiH, SekiguchiM (1992) MutT protein specifically hydrolyses a potent mutagenic substrate for DNA synthesis. Nature 355 : 273–275.
9. FeigDI, SowersLC, LoebLA (1994) Reverse chemical mutagenesis: identification of the mutagenic lesions resulting from reactive oxygen species-mediated damage to DNA. Proc Natl Acad Sci U S A 91 : 6609–6613.
10. PurmalAA, KowYW, WallaceSS (1994) 5-Hydroxypyrimidine deoxynucleoside triphosphates are more efficiently incorporated into DNA by exonuclease-free Klenow fragment than 8-oxopurine deoxynucleoside triphosphates. Nucleic Acids Res 22 : 3930–3935.
11. FotiJJ, DevadossB, WinklerJA, CollinsJJ, WalkerGC (2012) Oxidation of the guanine nucleotide pool underlies cell death by bactericidal antibiotics. Science 336 : 315–319.
12. GutierrezA, LauretiL, CrussardS, AbidaH, Rodriguez-RojasA, et al. (2013) beta-lactam antibiotics promote bacterial mutagenesis via an RpoS-mediated reduction in replication fidelity. Nat Commun 4 : 1610.
13. BessmanMJ, FrickDN, O'HandleySF (1996) The MutT proteins or “Nudix” hydrolases, a family of versatile, widely distributed, “housecleaning” enzymes. J Biol Chem 271 : 25059–25062.
14. GalperinMY, MorozOV, WilsonKS, MurzinAG (2006) House cleaning, a part of good housekeeping. Mol Microbiol 59 : 5–19.
15. McLennanAG (2006) The Nudix hydrolase superfamily. Cell Mol Life Sci 63 : 123–143.
16. TaddeiF, HayakawaH, BoutonM, CirinesiA, MaticI, et al. (1997) Counteraction by MutT protein of transcriptional errors caused by oxidative damage. Science 278 : 128–130.
17. FujikawaK, KamiyaH, YakushijiH, FujiiY, NakabeppuY, et al. (1999) The oxidized forms of dATP are substrates for the human MutT homologue, the hMTH1 protein. J Biol Chem 274 : 18201–18205.
18. TsuzukiT, EgashiraA, IgarashiH, IwakumaT, NakatsuruY, et al. (2001) Spontaneous tumorigenesis in mice defective in the MTH1 gene encoding 8-oxo-dGTPase. Proc Natl Acad Sci U S A 98 : 11456–11461.
19. NakabeppuY, OkaS, ShengZ, TsuchimotoD, SakumiK (2010) Programmed cell death triggered by nucleotide pool damage and its prevention by MutT homolog-1 (MTH1) with oxidized purine nucleoside triphosphatase. Mutat Res 703 : 51–58.
20. JarugaP, DizdarogluM (1996) Repair of products of oxidative DNA base damage in human cells. Nucleic Acids Res 24 : 1389–1394.
21. Murata-KamiyaN, KamiyaH, MuraokaM, KajiH, KasaiH (1997) Comparison of oxidation products from DNA components by gamma-irradiation and Fenton-type reactions. J Radiat Res 38 : 121–131.
22. FujikawaK, KamiyaH, KasaiH (1998) The mutations induced by oxidatively damaged nucleotides, 5-formyl-dUTP and 5-hydroxy-dCTP,in Escherichia coli. Nucleic Acids Res 26 : 4582–4587.
23. FragaCG, ShigenagaMK, ParkJW, DeganP, AmesBN (1990) Oxidative damage to DNA during aging: 8-hydroxy-2′-deoxyguanosine in rat organ DNA and urine. Proc Natl Acad Sci U S A 87 : 4533–4537.
24. HatahetZ, KowYW, PurmalAA, CunninghamRP, WallaceSS (1994) New substrates for old enzymes. 5-Hydroxy-2′-deoxycytidine and 5-hydroxy-2′-deoxyuridine are substrates for Escherichia coli endonuclease III and formamidopyrimidine DNA N-glycosylase, while 5-hydroxy-2′-deoxyuridine is a substrate for uracil DNA N-glycosylase. J Biol Chem 269 : 18814–18820.
25. PurmalAA, LampmanGW, BondJP, HatahetZ, WallaceSS (1998) Enzymatic processing of uracil glycol, a major oxidative product of DNA cytosine. J Biol Chem 273 : 10026–10035.
26. D'HamC, RomieuA, JaquinodM, GasparuttoD, CadetJ (1999) Excision of 5,6-dihydroxy-5,6-dihydrothymine, 5,6-dihydrothymine, and 5-hydroxycytosine from defined sequence oligonucleotides by Escherichia coli endonuclease III and Fpg proteins: kinetic and mechanistic aspects. Biochemistry 38 : 3335–3344.
27. MorozOV, MurzinAG, MakarovaKS, KooninEV, WilsonKS, et al. (2005) Dimeric dUTPases, HisE, and MazG belong to a new superfamily of all-alpha NTP pyrophosphohydrolases with potential “house-cleaning” functions. J Mol Biol 347 : 243–255.
28. LuLD, SunQ, FanXY, ZhongY, YaoYF, et al. (2010) Mycobacterial MazG is a novel NTP pyrophosphohydrolase involved in oxidative stress response. J Biol Chem 285 : 28076–28085.
29. GrossM, MarianovskyI, GlaserG (2006) MazG — a regulator of programmed cell death in Escherichia coli. Mol Microbiol 59 : 590–601.
30. NonakaM, TsuchimotoD, SakumiK, NakabeppuY (2009) Mouse RS21-C6 is a mammalian 2′-deoxycytidine 5′-triphosphate pyrophosphohydrolase that prefers 5-iodocytosine. Febs J 276 : 1654–1666.
31. WuB, LiuY, ZhaoQ, LiaoS, ZhangJ, et al. (2007) Crystal structure of RS21-C6, involved in nucleoside triphosphate pyrophosphohydrolysis. J Mol Biol 367 : 1405–1412.
32. ImlayJA, ChinSM, LinnS (1988) Toxic DNA damage by hydrogen peroxide through the Fenton reaction in vivo and in vitro. Science 240 : 640–642.
33. BoshoffHI, ReedMB, BarryCE3rd, MizrahiV (2003) DnaE2 polymerase contributes to in vivo survival and the emergence of drug resistance in Mycobacterium tuberculosis. Cell 113 : 183–193.
34. Saint-RufC, PesutJ, SoptaM, MaticI (2007) Causes and consequences of DNA repair activity modulation during stationary phase in Escherichia coli. Crit Rev Biochem Mol Biol 42 : 259–270.
35. MichaelsML, CruzC, GrollmanAP, MillerJH (1992) Evidence that MutY and MutM combine to prevent mutations by an oxidatively damaged form of guanine in DNA. Proc Natl Acad Sci U S A 89 : 7022–7025.
36. InoueM, KamiyaH, FujikawaK, OotsuyamaY, Murata-KamiyaN, et al. (1998) Induction of chromosomal gene mutations in Escherichia coli by direct incorporation of oxidatively damaged nucleotides. New evaluation method for mutagenesis by damaged DNA precursors in vivo. J Biol Chem 273 : 11069–11074.
37. HeepM, BrandstatterB, RiegerU, LehnN, RichterE, et al. (2001) Frequency of rpoB mutations inside and outside the cluster I region in rifampin-resistant clinical Mycobacterium tuberculosis isolates. J Clin Microbiol 39 : 107–110.
38. SchaaperRM, DanforthBN, GlickmanBW (1986) Mechanisms of spontaneous mutagenesis: an analysis of the spectrum of spontaneous mutation in the Escherichia coli lacI gene. J Mol Biol 189 : 273–284.
39. SchaaperRM, DunnRL (1991) Spontaneous mutation in the Escherichia coli lacI gene. Genetics 129 : 317–326.
40. SatouK, HarashimaH, KamiyaH (2003) Mutagenic effects of 2-hydroxy-dATP on replication in a HeLa extract: induction of substitution and deletion mutations. Nucleic Acids Res 31 : 2570–2575.
41. ZhengH, LuL, WangB, PuS, ZhangX, et al. (2008) Genetic basis of virulence attenuation revealed by comparative genomic analysis of Mycobacterium tuberculosis strain H37Ra versus H37Rv. PLoS One 3: e2375.
42. DarwinKH, NathanCF (2005) Role for nucleotide excision repair in virulence of Mycobacterium tuberculosis. Infect Immun 73 : 4581–4587.
43. DarwinKH, EhrtS, Gutierrez-RamosJC, WeichN, NathanCF (2003) The proteasome of Mycobacterium tuberculosis is required for resistance to nitric oxide. Science 302 : 1963–1966.
44. VenugopalA, BrykR, ShiS, RheeK, RathP, et al. (2011) Virulence of Mycobacterium tuberculosis depends on lipoamide dehydrogenase, a member of three multienzyme complexes. Cell Host Microbe 9 : 21–31.
45. ChanJ, XingY, MagliozzoRS, BloomBR (1992) Killing of virulent Mycobacterium tuberculosis by reactive nitrogen intermediates produced by activated murine macrophages. J Exp Med 175 : 1111–1122.
46. KamiyaH (2003) Mutagenic potentials of damaged nucleic acids produced by reactive oxygen/nitrogen species: approaches using synthetic oligonucleotides and nucleotides: survey and summary. Nucleic Acids Res 31 : 517–531.
47. Dos VultosT, BlazquezJ, RauzierJ, MaticI, GicquelB (2006) Identification of Nudix hydrolase family members with an antimutator role in Mycobacterium tuberculosis and Mycobacterium smegmatis. J Bacteriol 188 : 3159–3161.
48. PatilAG, SangPB, GovindanA, VarshneyU (2013) 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. J Biol Chem 288 : 11252–11262.
49. SangPB, VarshneyU (2013) Biochemical properties of MutT2 proteins from Mycobacterium tuberculosis and M. smegmatis and their contrasting antimutator roles in Escherichia coli. J Bacteriol 195 : 1552–1560.
50. KreutzerDA, EssigmannJM (1998) Oxidized, deaminated cytosines are a source of C —> T transitions in vivo. Proc Natl Acad Sci U S A 95 : 3578–3582.
51. BebenekK, PedersenLC, KunkelTA (2011) Replication infidelity via a mismatch with Watson-Crick geometry. Proc Natl Acad Sci U S A 108 : 1862–1867.
52. BjedovI, TenaillonO, GerardB, SouzaV, DenamurE, et al. (2003) Stress-induced mutagenesis in bacteria. Science 300 : 1404–1409.
53. IoergerTR, KooS, NoEG, ChenX, LarsenMH, et al. (2009) Genome analysis of multi - and extensively-drug-resistant tuberculosis from KwaZulu-Natal, South Africa. PLoS One 4: e7778.
54. FordCB, LinPL, ChaseMR, ShahRR, IartchoukO, et al. (2011) Use of whole genome sequencing to estimate the mutation rate of Mycobacterium tuberculosis during latent infection. Nat Genet 43 : 482–486.
55. SchapiroJM, LibbySJ, FangFC (2003) Inhibition of bacterial DNA replication by zinc mobilization during nitrosative stress. Proc Natl Acad Sci U S A 100 : 8496–8501.
56. LepoivreM, FieschiF, CovesJ, ThelanderL, FontecaveM (1991) Inactivation of ribonucleotide reductase by nitric oxide. Biochem Biophys Res Commun 179 : 442–448.
57. BarryCE3rd, BoshoffHI, DartoisV, DickT, EhrtS, et al. (2009) The spectrum of latent tuberculosis: rethinking the biology and intervention strategies. Nat Rev Microbiol 7 : 845–855.
58. DuttaNK, MehraS, DidierPJ, RoyCJ, DoyleLA, et al. (2010) Genetic requirements for the survival of tubercle bacilli in primates. J Infect Dis 201 : 1743–1752.
59. FritzC, MaassS, KreftA, BangeFC (2002) Dependence of Mycobacterium bovis BCG on anaerobic nitrate reductase for persistence is tissue specific. Infect Immun 70 : 286–291.
60. SinghR, RaoV, ShakilaH, GuptaR, KheraA, et al. (2003) Disruption of mptpB impairs the ability of Mycobacterium tuberculosis to survive in guinea pigs. Mol Microbiol 50 : 751–762.
61. MalhotraV, SharmaD, RamanathanVD, ShakilaH, SainiDK, et al. (2004) Disruption of response regulator gene, devR, leads to attenuation in virulence of Mycobacterium tuberculosis. FEMS Microbiol Lett 231 : 237–245.
62. DharN, McKinneyJD (2010) Mycobacterium tuberculosis persistence mutants identified by screening in isoniazid-treated mice. Proc Natl Acad Sci U S A 107 : 12275–12280.
63. KohanskiMA, DwyerDJ, HayeteB, LawrenceCA, CollinsJJ (2007) A common mechanism of cellular death induced by bactericidal antibiotics. Cell 130 : 797–810.
64. WangX, ZhaoX (2009) Contribution of oxidative damage to antimicrobial lethality. Antimicrob Agents Chemother 53 : 1395–1402.
65. GrantSS, KaufmannBB, ChandNS, HaseleyN, HungDT (2012) Eradication of bacterial persisters with antibiotic-generated hydroxyl radicals. Proc Natl Acad Sci U S A 109 : 12147–12152.
66. BardarovS, Bardarov JrSJr, Pavelka JrMSJr, SambandamurthyV, LarsenM, et al. (2002) Specialized transduction: an efficient method for generating marked and unmarked targeted gene disruptions in Mycobacterium tuberculosis, M. bovis BCG and M. smegmatis. Microbiology 148 : 3007–3017.
67. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed. , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
68. WalkerJM (1994) The bicinchoninic acid (BCA) assay for protein quantitation. Methods Mol Biol 32 : 5–8.
69. Van KesselJC, HatfullGF (2008) Mycobacterial recombineering. Methods Mol Biol 435 : 203–215.
70. VoskuilMI, SchnappingerD, ViscontiKC, HarrellMI, DolganovGM, et al. (2003) Inhibition of respiration by nitric oxide induces a Mycobacterium tuberculosis dormancy program. J Exp Med 198 : 705–713.
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