#PAGE_PARAMS# #ADS_HEAD_SCRIPTS# #MICRODATA#

Unexpected Role for DNA Polymerase I As a Source of Genetic Variability


Helicobacter pylori, a human pathogen infecting about half of the world population, is characterised by its large intraspecies variability. Its genome plasticity has been invoked as the basis for its high adaptation capacity. Consistent with its small genome, H. pylori possesses only two bona fide DNA polymerases, Pol I and the replicative Pol III, lacking homologues of translesion synthesis DNA polymerases. Bacterial DNA polymerases I are implicated both in normal DNA replication and in DNA repair. We report that H. pylori DNA Pol I 5′- 3′ exonuclease domain is essential for viability, probably through its involvement in DNA replication. We show here that, despite the fact that it also plays crucial roles in DNA repair, Pol I contributes to genomic instability. Indeed, strains defective in the DNA polymerase activity of the protein, although sensitive to genotoxic agents, display reduced mutation frequencies. Conversely, overexpression of Pol I leads to a hypermutator phenotype. Although the purified protein displays an intrinsic fidelity during replication of undamaged DNA, it lacks a proofreading activity, allowing it to efficiently elongate mismatched primers and perform mutagenic translesion synthesis. In agreement with this finding, we show that the spontaneous mutator phenotype of a strain deficient in the removal of oxidised pyrimidines from the genome is in part dependent on the presence of an active DNA Pol I. This study provides evidence for an unexpected role of DNA polymerase I in generating genomic plasticity.


Vyšlo v časopise: Unexpected Role for DNA Polymerase I As a Source of Genetic Variability. PLoS Genet 7(6): e32767. doi:10.1371/journal.pgen.1002152
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pgen.1002152

Souhrn

Helicobacter pylori, a human pathogen infecting about half of the world population, is characterised by its large intraspecies variability. Its genome plasticity has been invoked as the basis for its high adaptation capacity. Consistent with its small genome, H. pylori possesses only two bona fide DNA polymerases, Pol I and the replicative Pol III, lacking homologues of translesion synthesis DNA polymerases. Bacterial DNA polymerases I are implicated both in normal DNA replication and in DNA repair. We report that H. pylori DNA Pol I 5′- 3′ exonuclease domain is essential for viability, probably through its involvement in DNA replication. We show here that, despite the fact that it also plays crucial roles in DNA repair, Pol I contributes to genomic instability. Indeed, strains defective in the DNA polymerase activity of the protein, although sensitive to genotoxic agents, display reduced mutation frequencies. Conversely, overexpression of Pol I leads to a hypermutator phenotype. Although the purified protein displays an intrinsic fidelity during replication of undamaged DNA, it lacks a proofreading activity, allowing it to efficiently elongate mismatched primers and perform mutagenic translesion synthesis. In agreement with this finding, we show that the spontaneous mutator phenotype of a strain deficient in the removal of oxidised pyrimidines from the genome is in part dependent on the presence of an active DNA Pol I. This study provides evidence for an unexpected role of DNA polymerase I in generating genomic plasticity.


Zdroje

1. SuerbaumSMichettiP 2002 Helicobacter pylori infection. N Engl J Med 347 1175 1186

2. FalushDKraftCTaylorNSCorreaPFoxJG 2001 Recombination and mutation during long-term gastric colonization by Helicobacter pylori: estimates of clock rates, recombination size, and minimal age. Proc Natl Acad Sci U S A 98 15056 15061

3. IsraelDASalamaNKrishnaURiegerUMAthertonJC 2001 Helicobacter pylori genetic diversity within the gastric niche of a single human host. Proc Natl Acad Sci U S A 98 14625 14630

4. KangJBlaserMJ 2006 Bacterial populations as perfect gases: genomic integrity and diversification tensions in Helicobacter pylori. Nat Rev Microbiol 4 826 836

5. TenaillonOToupanceBLe NagardHTaddeiFGodelleB 1999 Mutators, population size, adaptive landscape and the adaptation of asexual populations of bacteria. Genetics 152 485 493

6. BjorkholmBSjolundMFalkPGBergOGEngstrandL 2001 Mutation frequency and biological cost of antibiotic resistance in Helicobacter pylori. Proc Natl Acad Sci U S A 98 14607 14612

7. PintoAVMathieuAMarsinSVeauteXIelpiL 2005 Suppression of homologous and homeologous recombination by the bacterial MutS2 protein. Mol Cell 17 113 120

8. KangJMIovineNMBlaserMJ 2006 A paradigm for direct stress-induced mutation in prokaryotes. FASEB J 20 2476 2485

9. O'RourkeEJChevalierCPintoAVThibergeJMIelpiL 2003 Pathogen DNA as target for host-generated oxidative stress: role for repair of bacterial DNA damage in Helicobacter pylori colonization. Proc Natl Acad Sci U S A 100 2789 2794

10. TippinBPhamPGoodmanMF 2004 Error-prone replication for better or worse. Trends Microbiol 12 288 295

11. KornbergABakerTA 1992 DNA Replication New York Freeman

12. JoyceCMGrindleyND 1984 Method for determining whether a gene of Escherichia coli is essential: application to the polA gene. J Bacteriol 158 636 643

13. OkazakiRArisawaMSuginoA 1971 Slow joining of newly replicated DNA chains in DNA polymerase I-deficient Escherichia coli mutants. Proc Natl Acad Sci U S A 68 2954 2957

14. AlmRALingLSMoirDTKingBLBrownED 1999 Genomic-sequence comparison of two unrelated isolates of the human gastric pathogen Helicobacter pylori. Nature 397 176 180

15. TombJFWhiteOKerlavageARClaytonRASuttonGG 1997 The complete genome sequence of the gastric pathogen Helicobacter pylori. Nature 388 539 547

16. DerbyshireVFreemontPSSandersonMRBeeseLFriedmanJM 1988 Genetic and crystallographic studies of the 3′,5′-exonucleolytic site of DNA polymerase I. Science 240 199 201

17. DerbyshireVPinsonneaultJKJoyceCM 1995 Structure-function analysis of 3′→5′-exonuclease of DNA polymerases. Methods Enzymol 262 363 385

18. LiuXHouJLiuJ 2006 Chlamydial DNA polymerase I can bypass lesions in vitro. Biochem Biophys Res Commun 345 1083 1091

19. ClarkJMBeardsleyGP 1989 Template length, sequence context, and 3′-5′ exonuclease activity modulate replicative bypass of thymine glycol lesions in vitro. Biochemistry 28 775 779

20. MathieuAO'RourkeEJRadicellaJP 2006 Helicobacter pylori genes involved in avoidance of mutations induced by 8-oxoguanine. J Bacteriol 188 7464 7469

21. JeongJYMukhopadhyayAKAkadaJKDailidieneDHoffmanPS 2001 Roles of FrxA and RdxA nitroreductases of Helicobacter pylori in susceptibility and resistance to metronidazole. J Bacteriol 183 5155 5162

22. BebenekKKunkelTA 1995 Analyzing fidelity of DNA polymerases. Methods Enzymol 262 217 232

23. BebenekKJoyceCMFitzgeraldMPKunkelTA 1990 The fidelity of DNA synthesis catalyzed by derivatives of Escherichia coli DNA polymerase I. J Biol Chem 265 13878 13887

24. EckertKAKunkelTA 1990 High fidelity DNA synthesis by the Thermus aquaticus DNA polymerase. Nucleic Acids Res 18 3739 3744

25. LongleyMJNguyenDKunkelTACopelandWC 2001 The fidelity of human DNA polymerase gamma with and without exonucleolytic proofreading and the p55 accessory subunit. J Biol Chem 276 38555 38562

26. IdeHKowYWWallaceSS 1985 Thymine glycols and urea residues in M13 DNA constitute replicative blocks in vitro. Nucleic Acids Res 13 8035 8052

27. StraussBRabkinSSagherDMooreP 1982 The role of DNA polymerase in base substitution mutagenesis on non-instructional templates. Biochimie 64 829 838

28. GrollmanAPMoriyaM 1993 Mutagenesis by 8-oxoguanine: an enemy within. Trends Genet 9 246 249

29. SuerbaumSJosenhansC 2007 Helicobacter pylori evolution and phenotypic diversification in a changing host. Nat Rev Microbiol 5 441 452

30. BatesHRandallSKRayssiguierCBridgesBAGoodmanMF 1989 Spontaneous and UV-induced mutations in Escherichia coli K-12 strains with altered or absent DNA polymerase I. J Bacteriol 171 2480 2484

31. LindahlT 1993 Instability and decay of the primary structure of DNA [see comments]. Nature 362 709 715

32. DuigouSEhrlichSDNoirotPNoirot-GrosMF 2005 DNA polymerase I acts in translesion synthesis mediated by the Y-polymerases in Bacillus subtilis. Mol Microbiol 57 678 690

33. O'RourkeEJChevalierCBoiteuxSLabigneAIelpiL 2000 A novel 3-methyladenine DNA glycosylase from helicobacter pylori defines a new class within the endonuclease III family of base excision repair glycosylases. J Biol Chem 275 20077 20083

34. EutseyRWangGMaierRJ 2007 Role of a MutY DNA glycosylase in combating oxidative DNA damage in Helicobacter pylori. DNA Repair (Amst) 6 19 26

35. KieferJRMaoCBramanJCBeeseLS 1998 Visualizing DNA replication in a catalytically active Bacillus DNA polymerase crystal. Nature 391 304 307

36. LiYKorolevSWaksmanG 1998 Crystal structures of open and closed forms of binary and ternary complexes of the large fragment of Thermus aquaticus DNA polymerase I: structural basis for nucleotide incorporation. EMBO J 17 7514 7525

37. BellJBEckertKAJoyceCMKunkelTA 1997 Base miscoding and strand misalignment errors by mutator Klenow polymerases with amino acid substitutions at tyrosine 766 in the O helix of the fingers subdomain. J Biol Chem 272 7345 7351

38. LoneSRomanoLJ 2003 Mechanistic insights into replication across from bulky DNA adducts: a mutant polymerase I allows an N-acetyl-2-aminofluorene adduct to be accommodated during DNA synthesis. Biochemistry 42 3826 3834

39. RechkoblitOKolbanovskiyAMalininaLGeacintovNEBroydeS 2010 Mechanism of error-free and semitargeted mutagenic bypass of an aromatic amine lesion by Y-family polymerase Dpo4. Nat Struct Mol Biol 17 379 388

40. WashingtonMTPrakashLPrakashS 2001 Yeast DNA polymerase eta utilizes an induced-fit mechanism of nucleotide incorporation. Cell 107 917 927

41. IdeHPetrulloLAHatahetZWallaceSS 1991 Processing of DNA base damage by DNA polymerases. Dihydrothymine and beta-ureidoisobutyric acid as models for instructive and noninstructive lesions. J Biol Chem 266 1469 1477

42. MatrayTJHaxtonKJGreenbergMM 1995 The effects of the ring fragmentation product of thymidine C5-hydrate on phosphodiesterases and klenow (exo-) fragment. Nucleic Acids Res 23 4642 4648

43. Paz-ElizurTTakeshitaMLivnehZ 1997 Mechanism of bypass synthesis through an abasic site analog by DNA polymerase I. Biochemistry 36 1766 1773

44. SagherDStraussB 1983 Insertion of nucleotides opposite apurinic/apyrimidinic sites in deoxyribonucleic acid during in vitro synthesis: uniqueness of adenine nucleotides. Biochemistry 22 4518 4526

45. HatahetZZhouMReha-KrantzLJIdeHMorricalSW 1999 In vitro selection of sequence contexts which enhance bypass of abasic sites and tetrahydrofuran by T4 DNA polymerase holoenzyme. J Mol Biol 286 1045 1057

46. PurmalAALampmanGWBondJPHatahetZWallaceSS 1998 Enzymatic processing of uracil glycol, a major oxidative product of DNA cytosine. J Biol Chem 273 10026 10035

47. ShibutaniSGrollmanAP 1993 On the mechanism of frameshift (deletion) mutagenesis in vitro. J Biol Chem 268 11703 11710

48. AranaMESekiMWoodRDRogozinIBKunkelTA 2008 Low-fidelity DNA synthesis by human DNA polymerase theta. Nucleic Acids Res 36 3847 3856

49. AranaMETakataKGarcia-DiazMWoodRDKunkelTA 2007 A unique error signature for human DNA polymerase nu. DNA Repair (Amst) 6 213 223

50. SekiMMasutaniCYangLWSchuffertAIwaiS 2004 High-efficiency bypass of DNA damage by human DNA polymerase Q. EMBO J 23 4484 4494

51. FeigDISowersLCLoebLA 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

52. KreutzerDAEssigmannJM 1998 Oxidized, deaminated cytosines are a source of C→T transitions in vivo. Proc Natl Acad Sci U S A 95 3578 3582

53. NajranaTSaitoYUrakiFKuboKYamamotoK 2000 Spontaneous and osmium tetroxide-induced mutagenesis in an Escherichia coli strain deficient in both endonuclease III and endonuclease VIII. Mutagenesis 15 121 125

54. PurmalAAKowYWWallaceSS 1994 Major oxidative products of cytosine, 5-hydroxycytosine and 5-hydroxyuracil, exhibit sequence context-dependent mispairing in vitro. Nucleic Acids Res 22 72 78

55. 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

56. WallaceSS 2002 Biological consequences of free radical-damaged DNA bases. Free Radic Biol Med 33 1 14

57. Makiela-DzbenskaKJaszczurMBanach-OrlowskaMJonczykPSchaaperRM 2009 Role of Escherichia coli DNA polymerase I in chromosomal DNA replication fidelity. Mol Microbiol 74 1114 1127

58. TagoYImaiMIharaMAtofujiHNagataY 2005 Escherichia coli mutator (Delta)polA is defective in base mismatch correction: the nature of in vivo DNA replication errors. J Mol Biol 351 299 308

59. HeuermannDHaasR 1998 A stable shuttle vector system for efficient genetic complementation of Helicobacter pylori strains by transformation and conjugation. Mol Gen Genet 257 519 528

60. SkouloubrisSThibergeJMLabigneADe ReuseH 1998 The Helicobacter pylori UreI protein is not involved in urease activity but is essential for bacterial survival in vivo. Infect Immun 66 4517 4521

61. KangJHuangSBlaserMJ 2005 Structural and functional divergence of MutS2 from bacterial MutS1 and eukaryotic MSH4-MSH5 homologs. J Bacteriol 187 3528 3537

62. MarsinSMathieuAKortulewskiTGueroisRRadicellaJP 2008 Unveiling novel RecO distant orthologues involved in homologous recombination. PLoS Genet 4 e1000146 doi:10.1371/journal.pgen.1000146

63. KokoskaRJMcCullochSDKunkelTA 2003 The efficiency and specificity of apurinic/apyrimidinic site bypass by human DNA polymerase eta and Sulfolobus solfataricus Dpo4. J Biol Chem 278 50537 50545

Štítky
Genetika Reprodukčná medicína

Článok vyšiel v časopise

PLOS Genetics


2011 Číslo 6
Najčítanejšie tento týždeň
Najčítanejšie v tomto čísle
Kurzy

Zvýšte si kvalifikáciu online z pohodlia domova

Získaná hemofilie - Povědomí o nemoci a její diagnostika
nový kurz

Eozinofilní granulomatóza s polyangiitidou
Autori: doc. MUDr. Martina Doubková, Ph.D.

Všetky kurzy
Prihlásenie
Zabudnuté heslo

Zadajte e-mailovú adresu, s ktorou ste vytvárali účet. Budú Vám na ňu zasielané informácie k nastaveniu nového hesla.

Prihlásenie

Nemáte účet?  Registrujte sa

#ADS_BOTTOM_SCRIPTS#