Human and bacterial oxidative demethylases repair alkylation damage in both RNA and DNA

Abstract

Repair of DNA damage is essential for maintaining genome integrity, and repair deficiencies in mammals are associated with cancer, neurological disease and developmental defects1. Alkylation damage in DNA is repaired by at least three different mechanisms, including damage reversal by oxidative demethylation of 1-methyladenine and 3-methylcytosine by Escherichia coli AlkB2,3. By contrast, little is known about consequences and cellular handling of alkylation damage to RNA4. Here we show that two human AlkB homologues, hABH2 and hABH3, also are oxidative DNA demethylases and that AlkB and hABH3, but not hABH2, also repair RNA. Whereas AlkB and hABH3 prefer single-stranded nucleic acids, hABH2 acts more efficiently on double-stranded DNA. In addition, AlkB and hABH3 expressed in E. coli reactivate methylated RNA bacteriophage MS2 in vivo, illustrating the biological relevance of this repair activity and establishing RNA repair as a potentially important defence mechanism in living cells. The different catalytic properties and the different subnuclear localization patterns shown by the human homologues indicate that hABH2 and hABH3 have distinct roles in the cellular response to alkylation damage.

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Figure 1: AlkB and human homologues.
Figure 2: Removal of methylation damage from RNA and DNA by AlkB, hABH2 and hABH3.
Figure 3: Host cell reactivation of MMS-treated bacteriophages in E. coli HK82F′ (alkB) by episomally expressed AlkB, hABH2 and hABH3.
Figure 4: Subcellular localization of hABH2 and hABH3.

References

  1. 1

    Friedberg, E. C., Walker, G. C. & Siede, W. DNA Repair and Mutagenesis (ASM, Washington DC, 1995)

    Google Scholar 

  2. 2

    Falnes, P. Ø., Johansen, R. F. & Seeberg, E. AlkB-mediated oxidative demethylation reverses DNA damage in Escherichia coli. Nature 419, 178–182 (2002)

    ADS  CAS  Article  Google Scholar 

  3. 3

    Trewick, S. C., Henshaw, T. F., Hausinger, R. P., Lindahl, T. & Sedgwick, B. Oxidative demethylation by Escherichia coli AlkB directly reverts DNA base damage. Nature 419, 174–178 (2002)

    ADS  CAS  Article  Google Scholar 

  4. 4

    Singer, B. & Grunberger, D. Molecular Biology of Mutagens and Carcinogens (Plenum, New York, 1983)

    Google Scholar 

  5. 5

    Boiteux, S. & Laval, J. Mutagenesis by alkylating agents: coding properties for DNA polymerase of poly(dC) template containing 3-methylcytosine. Biochimie 64, 637–641 (1982)

    CAS  Article  Google Scholar 

  6. 6

    Saffhill, R. Differences in the promutagenic nature of 3-methylcytosine as revealed by DNA and RNA polymerising enzymes. Carcinogenesis 5, 691–693 (1984)

    CAS  Article  Google Scholar 

  7. 7

    Rozenski, J., Crain, P. F. & McCloskey, J. A. The RNA Modification Database: 1999 update. Nucleic Acids Res. 27, 196–197 (1999)

    CAS  Article  Google Scholar 

  8. 8

    Agris, P. F. The importance of being modified: roles of modified nucleosides and Mg2+ in RNA structure and function. Prog. Nucleic Acid Res. Mol. Biol. 53, 79–129 (1996)

    CAS  Article  Google Scholar 

  9. 9

    Yoshizawa, S., Fourmy, D. & Puglisi, J. D. Recognition of the codon–anticodon helix by ribosomal RNA. Science 285, 1722–1725 (1999)

    CAS  Article  Google Scholar 

  10. 10

    Matsugi, J. & Murao, K. Study on construction of a cDNA library corresponding to an amino acid-specific tRNA and influence of the modified nucleotide upon nucleotide misincorporations in reverse transcription. Biochim. Biophys. Acta 1521, 81–88 (2001)

    CAS  Article  Google Scholar 

  11. 11

    Haug, T. et al. Human uracil-DNA glycosylase gene: sequence organization, methylation pattern, and mapping to chromosome 12q22–q24.1. Genomics 36, 408–416 (1996)

    CAS  Article  Google Scholar 

  12. 12

    Wei, Y. F., Carter, K. C., Wang, R. P. & Shell, B. K. Molecular cloning and functional analysis of a human cDNA encoding an Escherichia coli AlkB homolog, a protein involved in DNA alkylation damage repair. Nucleic Acids Res. 24, 931–937 (1996)

    CAS  Article  Google Scholar 

  13. 13

    Aravind, L. & Koonin, E. V. The DNA-repair protein AlkB, EGL-9, and leprecan define new families of 2-oxoglutarate- and iron-dependent dioxygenases. Genome Biol. 2, Research0007 [online] 〈http://genomebiology.com/2001/2/3/research/0007〉 (2001)

  14. 14

    Myllyharju, J. & Kivirikko, K. I. Characterization of the iron- and 2-oxoglutarate-binding sites of human prolyl 4-hydroxylase. EMBO J. 17, 1173–1180 (1997)

    Article  Google Scholar 

  15. 15

    Valegård, K. et al. Structure of a cephalosporin synthase. Nature 394, 805–809 (1998)

    ADS  Article  Google Scholar 

  16. 16

    Nash, T. The colorimetric estimation of formaldehyde by means of the Hantzsch reaction. Biochem. J. 55, 416–421 (1953)

    CAS  Article  Google Scholar 

  17. 17

    Kaasen, I., Evensen, G. & Seeberg, E. Amplified expression of the tag+ and alkA+ genes in Escherichia coli: identification of gene products and effects on alkylation resistance. J. Bacteriol. 168, 642–647 (1986)

    CAS  Article  Google Scholar 

  18. 18

    Leonhardt, H. et al. Dynamics of DNA replication factories in living cells. J. Cell Biol. 149, 271–280 (2000)

    CAS  Article  Google Scholar 

  19. 19

    Warner, J. R. Nascent ribosomes. Cell 107, 133–136 (2001)

    CAS  Article  Google Scholar 

  20. 20

    Hartl, F. U. & Hayer-Hartl, M. Molecular chaperones in the cytosol: from nascent chain to folded protein. Science 295, 1852–1858 (2002)

    ADS  CAS  Article  Google Scholar 

  21. 21

    Sambrook, J., Fritsch, E. F. & Maniatis, T. Molecular Cloning. A Laboratory Manual, 2nd edn (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989)

    Google Scholar 

  22. 22

    Yanisch-Perron, C., Vieira, J. & Messing, J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33, 103–119 (1985)

    CAS  Article  Google Scholar 

  23. 23

    Bjelland, S., Bjørås, M. & Seeberg, E. Excision of 3-methylguanine from alkylated DNA by 3-methyladenine DNA glycosylase I of Escherichia coli. Nucleic Acids Res. 21, 2045–2049 (1993)

    CAS  Article  Google Scholar 

  24. 24

    Huang, X., Powell, J., Mooney, L. A., Li, C. & Frenkel, K. Importance of complete DNA digestion in minimizing variability of 8-oxo-dG analyses. Free Radicals Biol. Med. 31, 1341–1351 (2001)

    CAS  Article  Google Scholar 

  25. 25

    Crain, P. F. Preparation and enzymatic hydrolysis of DNA and RNA for mass spectrometry. Methods Enzymol. 193, 782–790 (1990)

    CAS  Article  Google Scholar 

  26. 26

    Blatny, J. M., Brautaset, T., Winther-Larsen, H. C., Haugan, K. & Valla, S. Construction and use of a versatile set of broad-host-range cloning and expression vectors based on the RK2 replicon. Appl. Environ. Microbiol. 63, 370–379 (1997)

    CAS  PubMed  PubMed Central  Google Scholar 

  27. 27

    Dinglay, S., Trewick, S. C., Lindahl, T. & Sedgwick, B. Defective processing of methylated single-stranded DNA by E. coli AlkB mutants. Genes Dev. 14, 2097–2105 (2000)

    CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank E. Feyzi, K. Baynton and B. Kavli for comments; and A. Nilsen, M. Westbye, R. F. Johansen and L. Hagen for technical support. This work was supported by The Research Council of Norway and the Norwegian Cancer Society. H.E.K. acknowledges support from The Cancer Research Fund at the Regional Hospital in Trondheim, and Svanhild and Arne Must's Fund for Medical Research. E.S. acknowledges support from the European Commission and Simon Fougner Hartmann's Family Fund.

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Correspondence to Hans E. Krokan.

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Aas, P., Otterlei, M., Falnes, P. et al. Human and bacterial oxidative demethylases repair alkylation damage in both RNA and DNA. Nature 421, 859–863 (2003). https://doi.org/10.1038/nature01363

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