Abstract

DNA repair is essential to prevent the cytotoxic or mutagenic effects of various types of DNA lesions, which are sensed by distinct pathways to recruit repair factors specific to the damage type. Although biochemical mechanisms for repairing several forms of genomic insults are well understood, the upstream signalling pathways that trigger repair are established for only certain types of damage, such as double-stranded breaks and interstrand crosslinks1,2,3. Understanding the upstream signalling events that mediate recognition and repair of DNA alkylation damage is particularly important, since alkylation chemotherapy is one of the most widely used systemic modalities for cancer treatment and because environmental chemicals may trigger DNA alkylation4,5,6. Here we demonstrate that human cells have a previously unrecognized signalling mechanism for sensing damage induced by alkylation. We find that the alkylation repair complex ASCC (activating signal cointegrator complex)7 relocalizes to distinct nuclear foci specifically upon exposure of cells to alkylating agents. These foci associate with alkylated nucleotides, and coincide spatially with elongating RNA polymerase II and splicing components. Proper recruitment of the repair complex requires recognition of K63-linked polyubiquitin by the CUE (coupling of ubiquitin conjugation to ER degradation) domain of the subunit ASCC2. Loss of this subunit impedes alkylation adduct repair kinetics and increases sensitivity to alkylating agents, but not other forms of DNA damage. We identify RING finger protein 113A (RNF113A) as the E3 ligase responsible for upstream ubiquitin signalling in the ASCC pathway. Cells from patients with X-linked trichothiodystrophy, which harbour a mutation in RNF113A, are defective in ASCC foci formation and are hypersensitive to alkylating agents. Together, our work reveals a previously unrecognized ubiquitin-dependent pathway induced specifically to repair alkylation damage, shedding light on the molecular mechanism of X-linked trichothiodystrophy.

References

  1. Jackson, S. P.&Durocher, D.Regulation of DNA damage responses by ubiquitin and SUMO. Mol. Cell49, 795–807 (2013)

  2. Sirbu, B. M.&Cortez, D.DNA damage response: three levels of DNA repair regulation. Cold Spring Harb. Perspect. Biol.5, a012724 (2013)

  3. Zhao, Y., Brickner, J. R., Majid, M. C.&Mosammaparast, N.Crosstalk between ubiquitin and other post-translational modifications on chromatin during double-strand break repair. Trends Cell Biol.24, 426–434 (2014)

  4. Drabløs, F.et al.Alkylation damage in DNA and RNA—repair mechanisms and medical significance. DNA Repair3, 1389–1407 (2004)

  5. Fu, D., Calvo, J. A.&Samson, L. D.Balancing repair and tolerance of DNA damage caused by alkylating agents. Nat. Rev. Cancer12, 104–120 (2012)

  6. Sedgwick, B., Bates, P. A., Paik, J., Jacobs, S. C.&Lindahl, T.Repair of alkylatedDNA: recent advances. DNA Repair6, 429–442 (2007)

  7. Dango, S.et al.DNA unwinding by ASCC3 helicase is coupled to ALKBH3-dependent DNA alkylation repair and cancer cell proliferation. Mol. Cell44, 373–384 (2011)

  8. Wick, W.&Platten, M.Understanding and targeting alkylator resistance in glioblastoma. Cancer Discov.4, 1120–1122 (2014)

  9. Jung, D. J.et al.Novel transcription coactivator complex containing activating signal cointegrator 1. Mol. Cell. Biol.22, 5203–5211 (2002)

  10. Komander, D.&Rape, M.The ubiquitin code. Annu. Rev. Biochem.81, 203–229 (2012)

  11. Prag, G.et al.Mechanism of ubiquitin recognition by the CUE domain of Vps9p. Cell113, 609–620 (2003)

  12. Liu, S. et al.Promiscuous interactions of gp78 E3 ligase CUE domain with polyubiquitin chains. Structure20, 2138–2150 (2012)

  13. Unk, I.et al.Human SHPRH is a ubiquitin ligase for Mms2-Ubc13-dependent polyubiquitylation of proliferating cell nuclear antigen. Proc. Natl Acad. Sci. USA103, 18107–18112 (2006)

  14. Zhao, G. Y.et al.A critical role for the ubiquitin-conjugating enzyme Ubc13 in initiating homologous recombination. Mol. Cell25, 663–675 (2007)

  15. Thorslund, T.et al.Histone H1 couples initiation and amplification of ubiquitin signalling after DNA damage. Nature527, 389–393 (2015)

  16. Hegele, A.et al.Dynamic protein-protein interaction wiring of the human spliceosome. Mol. Cell45, 567–580 (2012)

  17. Corbett, M. A.et al.A novel X-linked trichothiodystrophy associated with a nonsense mutation in RNF113A. J. Med. Genet.52, 269–274 (2015)

  18. Nakabayashi, K.et al.Identification of C7orf11 (TTDN1) gene mutations and genetic heterogeneity in nonphotosensitive trichothiodystrophy. Am. J. Hum. Genet.76, 510–516 (2005)

  19. Sowa, M. E., Bennett, E. J., Gygi, S. P.&Harper, J. W.Defining the human deubiquitinating enzyme interaction landscape. Cell138, 389–403 (2009)

  20. Zhao, Y.et al.Noncanonical regulation of alkylation damage resistance by the OTUD4 deubiquitinase. EMBO J.34, 1687–1703 (2015)

  21. Eng, J. K., McCormack, A. L.&Yates, J. R.An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. J. Am. Soc. Mass Spectrom.5, 976–989 (1994)

Acknowledgements

We thank B. Sleckman, G. Oltz, T. Stappenbeck, S. Virgin, and K. Murphy for their advice on this manuscript. J.R.B. and A.K.B. are supported by a Cell and Molecular Biology Training Grant (5T32GM007067-40), and J.R.B. is also supported by a Shawn Hu and Angela Zeng Student Scholarship. J.M.S. is supported by a Monsanto Graduate Program Fellowship. P.M.L. is supported by a fellowship from the American Cancer Society (PF-14-182-01-DMC). We thank the patients and their families, whose help and participation made this work possible. We acknowledge the Alvin J. Siteman Cancer Center at Washington University and Barnes-Jewish Hospital for the use of the GEiC Core. The Siteman Cancer Center is supported by a National Cancer Institute Cancer Center Support Grant (P30 CA091842; Eberlein, PI). This work was supported by the National Institutes of Health (R01 GM108648 to A.V., R01 GM109102 to C.W., and R01 CA193318 to N.M.), the Alvin Siteman Cancer Research Fund, the Siteman Investment Program (both to N.M.), and the Children’s Discovery Institute of St. Louis Children’s Hospital (MC-II-2015-453 to N.M.).

Supplementary information

PDF files

  1. This file contains uncropped western blots and gels used in this study. Black rectangles denote how the blots and gels were cropped for final figures.

  2. All antibodies used in this study with concentrations noted. The antibodies were produced in either rabbit or mouse. Applications include Western blot (WB), immunofluorescence microscopy (IF), flow cytometry (FC) and proximity ligation assay (PLA).

Excel files

  1. Mass spectrometry data for TAP-ASCC2 purified from HeLa-S cells with or without prior exposure to MMS.

  2. A comprehensive list of each shRNA, including TRC numbers, used in the focused shRNA screen to identify the relevant E3 ligase.

  3. Mass spectrometry data for TAP-ASCC2 WT or the TAP-ASCC2 L506A CUE mutant purified from HeLa-S cells after MMS treatment.