A Germline Polymorphism of DNA Polymerase Beta Induces Genomic Instability and Cellular Transformation

English version České info

Several germline single nucleotide polymorphisms (SNPs) have been identified in the POLB gene, but little is known about their cellular and biochemical impact. DNA Polymerase β (Pol β), encoded by the POLB gene, is the main gap-filling polymerase involved in base excision repair (BER), a pathway that protects the genome from the consequences of oxidative DNA damage. In this study we tested the hypothesis that expression of the POLB germline coding SNP (rs3136797) in mammalian cells could induce a cancerous phenotype. Expression of this SNP in both human and mouse cells induced double-strand breaks, chromosomal aberrations, and cellular transformation. Following treatment with an alkylating agent, cells expressing this coding SNP accumulated BER intermediate substrates, including single-strand and double-strand breaks. The rs3136797 SNP encodes the P242R variant Pol β protein and biochemical analysis showed that P242R protein had a slower catalytic rate than WT, although P242R binds DNA similarly to WT. Our results suggest that people who carry the rs3136797 germline SNP may be at an increased risk for cancer susceptibility.

Vyšlo v časopise: A Germline Polymorphism of DNA Polymerase Beta Induces Genomic Instability and Cellular Transformation. PLoS Genet 8(11): e32767. doi:10.1371/journal.pgen.1003052
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pgen.1003052

Souhrn

Zdroje

1. LindahlT (1993) Instability and decay of the primary structure of DNA. Nature 362: 709–715.

2. BarnesDE, LindahlT (2004) Repair and genetic consequences of endogenous DNA base damage in mammalian cells. Annu Rev Genet 38: 445–476.

3. BeardWA, WilsonSH (2006) Structure and mechanism of DNA polymerase Beta. Chem Rev 106: 361–382.

4. SugoN, ArataniY, NagashimaY, KubotaY, KoyamaH (2000) Neonatal lethality with abnormal neurogenesis in mice deficient in DNA polymerase beta. Embo J 19: 1397–1404.

5. MohrenweiserHW, XiT, Vazquez-MatiasJ, JonesIM (2002) Identification of 127 amino acid substitution variants in screening 37 DNA repair genes in humans. Cancer Epidemiol Biomarkers Prev 11: 1054–1064.

6. YamtichJ, SpeedWC, StrakaE, KiddJR, SweasyJB, et al. (2009) Population-specific variation in haplotype composition and heterozygosity at the POLB locus. DNA Repair (Amst) 8: 579–584.

7. El-AndaloussiN, ValovkaT, ToueilleM, HassaPO, GehrigP, et al. (2007) Methylation of DNA polymerase beta by protein arginine methyltransferase 1 regulates its binding to proliferating cell nuclear antigen. Faseb J 21: 26–34.

8. GuoZ, ZhengL, DaiH, ZhouM, XuH, et al. (2009) Human DNA polymerase beta polymorphism, Arg137Gln, impairs its polymerase activity and interaction with PCNA and the cellular base excision repair capacity. Nucleic Acids Res 37: 3431–3441.

9. MatakidouA, el GaltaR, WebbEL, RuddMF, BridleH (2007) Genetic variation in the DNA repair genes is predictive of outcome in lung cancer. Hum Mol Genet 16: 2333–2340.

10. SellickGS, WadeR, RichardsS, OscierDG, CatovskyD, et al. (2008) Scan of 977 nonsynonymous SNPs in CLL4 trial patients for the identification of genetic variants influencing prognosis. Blood 111: 1625–1633.

11. LinGC, JaegerJ, EckertKA, SweasyJB (2009) Loop II of DNA polymerase beta is important for discrimination during substrate binding. DNA Repair (Amst) 8: 182–189.

12. LinGC, JaegerJ, SweasyJB (2007) Loop II of DNA polymerase beta is important for polymerization activity and fidelity. Nucleic Acids Res 35: 2924–2935.

13. SawayaMR, PrasadR, WilsonSH, KrautJ, PelletierH (1997) Crystal structures of human DNA polymerase beta complexed with gapped and nicked DNA: evidence for an induced fit mechanism. Biochemistry 36: 11205–11215.

14. SweasyJB, LangT, StarcevicD, SunKW, LaiCC, et al. (2005) Expression of DNA polymerase {beta} cancer-associated variants in mouse cells results in cellular transformation. Proc Natl Acad Sci U S A 102: 14350–14355.

15. LangT, DalalS, ChikovaA, DiMaioD, SweasyJB (2007) The E295K DNA polymerase beta gastric cancer-associated variant interferes with base excision repair and induces cellular transformation. Mol Cell Biol 27: 5587–5596.

16. LangT, MaitraM, StarcevicD, LiSX, SweasyJB (2004) A DNA polymerase beta mutant from colon cancer cells induces mutations. Proc Natl Acad Sci U S A 101: 6074–6079.

17. MaW, WestmorelandJW, GordeninDA, ResnickMA (2011) Alkylation base damage is converted into repairable double-strand breaks and complex intermediates in G2 cells lacking AP endonuclease. PLoS Genet 7: e1002059 doi:10.1371/journal.pgen.1002059

18. KosaJL, SweasyJB (1999) The E249K mutator mutant of DNA polymerase beta extends mispaired termini. J Biol Chem 274: 35866–35872.

19. DalalS, KosaJL, SweasyJB (2004) The D246V mutant of DNA polymerase beta misincorporates nucleotides: evidence for a role for the flexible loop in DNA positioning within the active site. J Biol Chem 279: 577–584.

20. Garcia-DiazM, BebenekK, KrahnJM, BlancoL, KunkelTA, et al. (2004) A structural solution for the DNA polymerase lambda-dependent repair of DNA gaps with minimal homology. Mol Cell 13: 561–572.

21. Garcia-DiazM, DominguezO, Lopez-FernandezLA, de LeraLT, SanigerML, et al. (2000) DNA polymerase lambda (Pol lambda), a novel eukaryotic DNA polymerase with a potential role in meiosis. J Mol Biol 301: 851–867.

22. OlivePL, BanathJP (2006) The comet assay: a method to measure DNA damage in individual cells. Nat Protoc 1: 23–29.

23. PascucciB, RussoMT, CrescenziM, BignamiM, DogliottiE (2005) The accumulation of MMS-induced single strand breaks in G1 phase is recombinogenic in DNA polymerase beta defective mammalian cells. Nucleic Acids Res 33: 280–288.

24. HeacockML, StefanickDF, HortonJK, WilsonSH (2010) Alkylation DNA damage in combination with PARP inhibition results in formation of S-phase-dependent double-strand breaks. DNA Repair (Amst) 9: 929–936.

25. MorenoV, GemignaniF, LandiS, Gioia-PatricolaL, ChabrierA, et al. (2006) Polymorphisms in genes of nucleotide and base excision repair: risk and prognosis of colorectal cancer. Clin Cancer Res 12: 2101–2108.

26. StarcevicD, DalalS, SweasyJ (2005) Hinge residue Ile260 of DNA polymerase beta is important for enzyme activity and fidelity. Biochemistry 44: 3775–3784.

27. JakubczakJL, MerlinoG, FrenchJE, MullerWJ, PaulB, et al. (1996) Analysis of genetic instability during mammary tumor progression using a novel selection-based assay for in vivo mutations in a bacteriophage lambda transgene target. Proc Natl Acad Sci U S A 93: 9073–9078.

28. GlazerPM, SarkarSN, SummersWC (1986) Detection and analysis of UV-induced mutations in mammalian cell DNA using a lambda phage shuttle vector. Proc Natl Acad Sci U S A 83: 1041–1044.

29. GuntherEJ, MurrayNE, GlazerPM (1993) High efficiency, restriction-deficient in vitro packaging extracts for bacteriophage lambda DNA using a new E.coli lysogen. Nucleic Acids Res 21: 3903–3904.

30. SobolRW, HortonJK, KuhnR, GuH, SinghalRK, et al. (1996) Requirement of mammalian DNA polymerase-beta in base-excision repair. Nature 379: 183–186.

31. SobolRW (2007) DNA polymerase beta null mouse embryonic fibroblasts harbor a homozygous null mutation in DNA polymerase iota. DNA Repair (Amst) 6: 3–7.

32. LowyDR, RandsE, ScolnickEM (1978) Helper-independent transformation by unintegrated Harvey sarcoma virus DNA. J Virol 26: 291–298.

33. OlivePL, BanathJP (2006) The comet assay: a method to measure DNA damage in individual cells. Nature protocols 1: 23–29.

34. MurphyDL, KosaJ, JaegerJ, SweasyJB (2008) The Asp285 variant of DNA polymerase beta extends mispaired primer termini via increased nucleotide binding. Biochemistry 47: 8048–8057.

35. MurphyDL, JaegerJ, SweasyJB (2011) A triad interaction in the fingers subdomain of DNA polymerase beta controls polymerase activity. J Am Chem Soc 133: 6279–6287.

36. YamtichJ, StarcevicD, LauperJ, SmithE, ShiI, et al. (2010) Hinge residue I174 is critical for proper dNTP selection by DNA polymerase beta. Biochemistry 49: 2326–2334.

37. DalalS, ChikovaA, JaegerJ, SweasyJB (2008) The Leu22Pro tumor-associated variant of DNA polymerase beta is dRP lyase deficient. Nucleic Acids Res 36: 411–422.