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A quantitative assay for assessing the effects of DNA lesions on transcription

Nature Chemical Biology volume 8, pages 817–822 (2012)Cite this article

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Abstract

Most mammalian cells in nature are quiescent but actively transcribing mRNA for normal physiological processes; thus, it is important to investigate how endogenous and exogenous DNA damage compromises transcription in cells. Here we describe a new competitive transcription and adduct bypass (CTAB) assay to determine the effects of DNA lesions on the fidelity and efficiency of transcription. Using this strategy, we demonstrate that the oxidatively induced lesions 8,5′-cyclo-2′-deoxyadenosine (cdA) and 8,5′-cyclo-2′-deoxyguanosine (cdG) and the methylglyoxal-induced lesion N2-(1-carboxyethyl)-2′-deoxyguanosine (N2-CEdG) strongly inhibited transcription in vitro and in mammalian cells. In addition, cdA and cdG, but not N2-CEdG, induced transcriptional mutagenesis in vitro and in vivo. Furthermore, when located on the template DNA strand, all examined lesions were primarily repaired by transcription-coupled nucleotide excision repair in mammalian cells. This newly developed CTAB assay should be generally applicable for quantitatively assessing how other DNA lesions affect DNA transcription in vitro and in cells.

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Figure 1: Chemical structures of lesions included in the present study.
Figure 2: A schematic diagram depicts the CTAB assay system.
Figure 3: Transcriptional alterations induced by S-cdA and S-cdG.
Figure 4: Transcriptional alterations induced by R- and S-N2-CEdG.

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References

  1. Brégeon, D. & Doetsch, P.W. Transcriptional mutagenesis: causes and involvement in tumour development. Nat. Rev. Cancer 11, 218–227 (2011).

    Article  Google Scholar 

  2. Lindahl, T. Instability and decay of the primary structure of DNA. Nature 362, 709–715 (1993).

    Article  CAS  Google Scholar 

  3. Saxowsky, T.T. & Doetsch, P.W. RNA polymerase encounters with DNA damage: transcription-coupled repair or transcriptional mutagenesis? Chem. Rev. 106, 474–488 (2006).

    Article  CAS  Google Scholar 

  4. Hanawalt, P.C. & Spivak, G. Transcription-coupled DNA repair: two decades of progress and surprises. Nat. Rev. Mol. Cell Biol. 9, 958–970 (2008).

    Article  CAS  Google Scholar 

  5. Kuraoka, I. & Tanaka, K. Assays for transcription elongation by RNA polymerase II using oligo(dC)-tailed template with single DNA damage. Methods Enzymol. 408, 214–223 (2006).

    Article  CAS  Google Scholar 

  6. Saxowsky, T.T., Meadows, K.L., Klungland, A. & Doetsch, P.W. 8-Oxoguanine-mediated transcriptional mutagenesis causes Ras activation in mammalian cells. Proc. Natl. Acad. Sci. USA 105, 18877–18882 (2008).

    Article  CAS  Google Scholar 

  7. Brégeon, D., Peignon, P.A. & Sarasin, A. Transcriptional mutagenesis induced by 8-oxoguanine in mammalian cells. PLoS Genet. 5, e1000577 (2009).

    Article  Google Scholar 

  8. Burns, J.A., Dreij, K., Cartularo, L. & Scicchitano, D.A. O6-methylguanine induces altered proteins at the level of transcription in human cells. Nucleic Acids Res. 38, 8178–8187 (2010).

    Article  CAS  Google Scholar 

  9. Brégeon, D., Doddridge, Z.A., You, H.J., Weiss, B. & Doetsch, P.W. Transcriptional mutagenesis induced by uracil and 8-oxoguanine in Escherichia coli. Mol. Cell 12, 959–970 (2003).

    Article  Google Scholar 

  10. Viswanathan, A., You, H.J. & Doetsch, P.W. Phenotypic change caused by transcriptional bypass of uracil in nondividing cells. Science 284, 159–162 (1999).

    Article  CAS  Google Scholar 

  11. Brooks, P.J. et al. The oxidative DNA lesion 8,5′-(S)-cyclo-2′-deoxyadenosine is repaired by the nucleotide excision repair pathway and blocks gene expression in mammalian cells. J. Biol. Chem. 275, 22355–22362 (2000).

    Article  CAS  Google Scholar 

  12. Marietta, C. & Brooks, P.J. Transcriptional bypass of bulky DNA lesions causes new mutant RNA transcripts in human cells. EMBO Rep. 8, 388–393 (2007).

    Article  CAS  Google Scholar 

  13. Kuraoka, I. et al. Removal of oxygen free-radical-induced 5′,8-purine cyclodeoxynucleosides from DNA by the nucleotide excision-repair pathway in human cells. Proc. Natl. Acad. Sci. USA 97, 3832–3837 (2000).

    Article  CAS  Google Scholar 

  14. Jaruga, P. & Dizdaroglu, M. 8,5′-Cyclopurine-2′-deoxynucleosides in DNA: mechanisms of formation, measurement, repair and biological effects. DNA Repair (Amst.) 7, 1413–1425 (2008).

    Article  CAS  Google Scholar 

  15. Belmadoui, N. et al. Radiation-induced formation of purine 5′,8-cyclonucleosides in isolated and cellular DNA: high stereospecificity and modulating effect of oxygen. Org. Biomol. Chem. 8, 3211–3219 (2010).

    Article  CAS  Google Scholar 

  16. Chatgilialoglu, C., Ferreri, C. & Terzidis, M.A. Purine 5′,8-cyclonucleoside lesions: chemistry and biology. Chem. Soc. Rev. 40, 1368–1382 (2011).

    Article  CAS  Google Scholar 

  17. Wang, J. et al. Quantification of oxidative DNA lesions in tissues of Long-Evans Cinnamon rats by capillary high-performance liquid chromatography-tandem mass spectrometry coupled with stable isotope-dilution method. Anal. Chem. 83, 2201–2209 (2011).

    Article  CAS  Google Scholar 

  18. D'Errico, M. et al. New functions of XPC in the protection of human skin cells from oxidative damage. EMBO J. 25, 4305–4315 (2006).

    Article  CAS  Google Scholar 

  19. Rodriguez, H. et al. Lymphoblasts of women with BRCA1 mutations are deficient in cellular repair of 8,5′-cyclopurine-2′-deoxynucleosides and 8-hydroxy-2′-deoxyguanosine. Biochemistry 46, 2488–2496 (2007).

    Article  CAS  Google Scholar 

  20. Thornalley, P.J. Pharmacology of methylglyoxal: formation, modification of proteins and nucleic acids, and enzymatic detoxification—a role in pathogenesis and antiproliferative chemotherapy. Gen. Pharmacol. 27, 565–573 (1996).

    Article  CAS  Google Scholar 

  21. Frischmann, M., Bidmon, C., Angerer, J. & Pischetsrieder, M. Identification of DNA adducts of methylglyoxal. Chem. Res. Toxicol. 18, 1586–1592 (2005).

    Article  CAS  Google Scholar 

  22. Schneider, M. et al. Determination of glycated nucleobases in human urine by a new monoclonal antibody specific for N2-carboxyethyl-2′-deoxyguanosine. Chem. Res. Toxicol. 17, 1385–1390 (2004).

    Article  CAS  Google Scholar 

  23. Li, H. et al. N2-carboxyethyl-2′-deoxyguanosine, a DNA glycation marker, in kidneys and aortas of diabetic and uremic patients. Kidney Int. 69, 388–392 (2006).

    Article  CAS  Google Scholar 

  24. Synold, T. et al. Advanced glycation end products of DNA: quantification of N2-(1-carboxyethyl)-2′-deoxyguanosine in biological samples by liquid chromatography electrospray ionization tandem mass spectrometry. Chem. Res. Toxicol. 21, 2148–2155 (2008).

    Article  CAS  Google Scholar 

  25. Yuan, B., Cao, H., Jiang, Y., Hong, H. & Wang, Y. Efficient and accurate bypass of N2-(1-carboxyethyl)-2′-deoxyguanosine by DinB DNA polymerase in vitro and in vivo. Proc. Natl. Acad. Sci. USA 105, 8679–8684 (2008).

    Article  CAS  Google Scholar 

  26. Murata-Kamiya, N., Kaji, H. & Kasai, H. Deficient nucleotide excision repair increases base-pair substitutions but decreases TGGC frameshifts induced by methylglyoxal in Escherichia coli. Mutat. Res. 442, 19–28 (1999).

    Article  CAS  Google Scholar 

  27. Tamae, D., Lim, P., Wuenschell, G.E. & Termini, J. Mutagenesis and repair induced by the DNA advanced glycation end product N2-1-(carboxyethyl)-2′-deoxyguanosine in human cells. Biochemistry 50, 2321–2329 (2011).

    Article  CAS  Google Scholar 

  28. Delaney, J.C. & Essigmann, J.M. Mutagenesis, genotoxicity, and repair of 1-methyladenine, 3-alkylcytosines, 1-methylguanine, and 3-methylthymine in alkB Escherichia coli. Proc. Natl. Acad. Sci. USA 101, 14051–14056 (2004).

    Article  CAS  Google Scholar 

  29. Delaney, J.C. & Essigmann, J.M. Assays for determining lesion bypass efficiency and mutagenicity of site-specific DNA lesions in vivo. Methods Enzymol. 408, 1–15 (2006).

    Article  CAS  Google Scholar 

  30. Masters, B.S., Stohl, L.L. & Clayton, D.A. Yeast mitochondrial RNA polymerase is homologous to those encoded by bacteriophages T3 and T7. Cell 51, 89–99 (1987).

    Article  CAS  Google Scholar 

  31. Yuan, B. et al. The roles of DNA polymerases κ and ι in the error-free bypass of N2-carboxyalkyl-2′-deoxyguanosine lesions in mammalian cells. J. Biol. Chem. 286, 17503–17511 (2011).

    Article  CAS  Google Scholar 

  32. Hong, H., Cao, H. & Wang, Y. Formation and genotoxicity of a guanine-cytosine intrastrand cross-link lesion in vivo. Nucleic Acids Res. 35, 7118–7127 (2007).

    Article  CAS  Google Scholar 

  33. Rolig, R.L. et al. Survival, mutagenesis, and host cell reactivation in a Chinese hamster ovary cell ERCC1 knock-out mutant. Mutagenesis 12, 277–283 (1997).

    Article  CAS  Google Scholar 

  34. Yuan, B., Wang, J., Cao, H., Sun, R. & Wang, Y. High-throughput analysis of the mutagenic and cytotoxic properties of DNA lesions by next-generation sequencing. Nucleic Acids Res. 39, 5945–5954 (2011).

    Article  CAS  Google Scholar 

  35. Clauson, C.L., Oestreich, K.J., Austin, J.W. & Doetsch, P.W. Abasic sites and strand breaks in DNA cause transcriptional mutagenesis in Escherichia coli. Proc. Natl. Acad. Sci. USA 107, 3657–3662 (2010).

    Article  CAS  Google Scholar 

  36. Clauson, C.L., Saxowsky, T.T. & Doetsch, P.W. Dynamic flexibility of DNA repair pathways in growth arrested Escherichia coli. DNA Repair (Amst.) 9, 842–847 (2010).

    Article  CAS  Google Scholar 

  37. Jasti, V.P. et al. (5′S)-8,5′-Cyclo-2′-deoxyguanosine is a strong block to replication, a potent pol V-dependent mutagenic lesion, and is inefficiently repaired in Escherichia coli. Biochemistry 50, 3862–3865 (2011).

    Article  CAS  Google Scholar 

  38. Kuraoka, I. et al. Oxygen free radical damage to DNA. Translesion synthesis by human DNA polymerase η and resistance to exonuclease action at cyclopurine deoxynucleoside residues. J. Biol. Chem. 276, 49283–49288 (2001).

    Article  CAS  Google Scholar 

  39. Tornaletti, S. Transcription arrest at DNA damage sites. Mutat. Res. 577, 131–145 (2005).

    Article  CAS  Google Scholar 

  40. Steitz, T.A. The structural changes of T7 RNA polymerase from transcription initiation to elongation. Curr. Opin. Struct. Biol. 19, 683–690 (2009).

    Article  CAS  Google Scholar 

  41. Kornberg, R. The molecular basis of eukaryotic transcription (Nobel Lecture). Angew. Chem. Int. Edn Engl. 46, 6956–6965 (2007).

    Article  CAS  Google Scholar 

  42. Brooks, P.J. The 8,5′-cyclopurine-2′-deoxynucleosides: candidate neurodegenerative DNA lesions in xeroderma pigmentosum, and unique probes of transcription and nucleotide excision repair. DNA Repair (Amst.) 7, 1168–1179 (2008).

    Article  CAS  Google Scholar 

  43. Choi, J.Y. & Guengerich, F.P. Kinetic evidence for inefficient and error-prone bypass across bulky N2-guanine DNA adducts by human DNA polymerase ι. J. Biol. Chem. 281, 12315–12324 (2006).

    Article  CAS  Google Scholar 

  44. Choi, J.Y., Angel, K.C. & Guengerich, F.P. Translesion synthesis across bulky N2-alkyl guanine DNA adducts by human DNA polymerase κ. J. Biol. Chem. 281, 21062–21072 (2006).

    Article  CAS  Google Scholar 

  45. Jarosz, D.F., Godoy, V.G., Delaney, J.C., Essigmann, J.M. & Walker, G.C. A single amino acid governs enhanced activity of DinB DNA polymerases on damaged templates. Nature 439, 225–228 (2006).

    Article  Google Scholar 

  46. Cohen, S.E. et al. Roles for the transcription elongation factor NusA in both DNA repair and damage tolerance pathways in Escherichia coli. Proc. Natl. Acad. Sci. USA 107, 15517–15522 (2010).

    Article  CAS  Google Scholar 

  47. Cheng, T.F., Hu, X., Gnatt, A. & Brooks, P.J. Differential blocking effects of the acetaldehyde-derived DNA lesion N2-ethyl-2′-deoxyguanosine on transcription by multisubunit and single subunit RNA polymerases. J. Biol. Chem. 283, 27820–27828 (2008).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank T.R. O'Connor (City of Hope), G.P. Pfeifer (City of Hope) and M. Seidman (National Institute of Aging) for providing cell lines and plasmid. This work was supported by the US National Institutes of Health (R01 DK082779, R01 ES019873 and R01 CA101864 to Y.W. and R01 ES016114 to L.J.N.).

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Authors and Affiliations

  1. Department of Chemistry, University of California–Riverside, Riverside, California, USA

    Changjun You, Xiaoxia Dai, Bifeng Yuan, Jin Wang, Jianshuang Wang & Yinsheng Wang

  2. Laboratory of Neurogenetics, National Institute on Alcohol Abuse and Alcoholism, Rockville, Maryland, USA

    Philip J Brooks

  3. Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA

    Laura J Niedernhofer

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Contributions

C.Y., X.D., B.Y. and Y.W. designed research; C.Y., X.D., B.Y. and Jianshuang Wang performed research; C.Y., X.D. and Y.W. analyzed data; C.Y., Jin Wang, P.J.B., L.J.N. and Y.W. wrote the paper; Y.W. conceived and supervised the study.

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Correspondence to Yinsheng Wang.

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You, C., Dai, X., Yuan, B. et al. A quantitative assay for assessing the effects of DNA lesions on transcription. Nat Chem Biol 8, 817–822 (2012). https://doi.org/10.1038/nchembio.1046

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