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A human MUTYH variant linking colonic polyposis to redox degradation of the [4Fe4S]2+ cluster

Abstract

The human DNA repair enzyme MUTYH excises mispaired adenine residues in oxidized DNA. Homozygous MUTYH mutations underlie the autosomal, recessive cancer syndrome MUTYH-associated polyposis. We report a MUTYH variant, p.C306W (c.918C>G), with a tryptophan residue in place of native cysteine, that ligates the [4Fe4S] cluster in a patient with colonic polyposis and family history of early age colon cancer. In bacterial MutY, the [4Fe4S] cluster is redox active, allowing rapid localization to target lesions by long-range, DNA-mediated signalling. In the current study, using DNA electrochemistry, we determine that wild-type MUTYH is similarly redox-active, but MUTYH C306W undergoes rapid oxidative degradation of its cluster to [3Fe4S]+, with loss of redox signalling. In MUTYH C306W, oxidative cluster degradation leads to decreased DNA binding and enzyme function. This study confirms redox activity in eukaryotic DNA repair proteins and establishes MUTYH C306W as a pathogenic variant, highlighting the essential role of redox signalling by the [4Fe4S] cluster.

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Fig. 1: A novel human MUTYH variant, C306W, lacks glycosylase activity.
Fig. 2: Initial electrochemical and spectroscopic characterization of MUTYH variants.
Fig. 3: Characterization of MUTYH in HEPES and analysis of the C306W decay product.

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References

  1. Markkanen, E., Dorn, J. & Hubscher, U. MUTYH DNA glycosylase: the rationale for removing undamaged bases from the DNA. Front. Genet. 4, 18 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Al-Tassan, N. et al. Inherited variants of MYH associated with somatic G:C→T:A mutations in colorectal tumors. Nat. Genet. 30, 227–232 (2002).

    Article  CAS  PubMed  Google Scholar 

  3. Sampson, J. R. et al. Autosomal recessive colorectal adenomatous polyposis due to inherited mutations of MYH. Lancet 362, 39–41 (2003).

    Article  CAS  PubMed  Google Scholar 

  4. Sieber, O. M. et al. Multiple colorectal adenomas, classic adenomatous polyposis, and germ-line mutations in MYH. N. Engl. J. Med. 348, 791–799 (2003).

    Article  Google Scholar 

  5. Cleary, S. P. et al. Germline MutY human homologue mutations and colorectal cancer: a multisite case–control study. Gastroenterology 136, 1251–1260 (2009).

    Article  CAS  PubMed  Google Scholar 

  6. Jones, N. et al. Increased colorectal cancer incidence in obligate carriers of heterozygous mutations in MUTYH. Gastroenterology 137, 489–494 (2009).

    Article  PubMed  Google Scholar 

  7. Out, A. A. et al. Leiden Open Variation Database of the MUTYH gene. Human Mutat. 31, 1205–1215 (2010).

    Article  CAS  Google Scholar 

  8. Maio, N. & Rouault, T. A. Iron–sulfur cluster biogenesis in mammalian cells: new insights into the molecular mechanisms of cluster delivery. Biochim. Biophys. Acta 1853, 1493–1512 (2015).

    Article  CAS  PubMed  Google Scholar 

  9. Alseth, I. et al. The Saccharomyces cerevisiae homologues of endonuclease III from Escherichia coli, Ntg1 and Ntg2, are both required for efficient repair of spontaneous and induced oxidative DNA damage in yeast. Mol. Cell Biol. 19, 3779–3787 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Trasvina-Arenas, C. H., Lopez-Castillo, L. M., Sanchez-Sandoval, E. & Brieba, L. G. Dispensability of the [4Fe-4S] cluster in novel homologues of adenine glycosylase MutY. FEBS J. 283, 521–540 (2016).

    Article  CAS  PubMed  Google Scholar 

  11. Cunningham, R. P. et al. Endonuclease III is an iron–sulfur protein. Biochemistry 28, 4450–4455 (1989).

    Article  CAS  PubMed  Google Scholar 

  12. Porello, S. L., Cannon, M. J. & David, S. S. A substrate recognition role for the [4Fe-4S]2+ cluster of the DNA repair glycosylase MutY. Biochemistry 37, 6465–6475 (1998).

    Article  CAS  PubMed  Google Scholar 

  13. Boal, A. K. et al. DNA-bound redox activity of DNA repair glycosylases containing [4Fe-4S] clusters. Biochemistry 44, 8397–8407 (2005).

    Article  CAS  PubMed  Google Scholar 

  14. Gorodetsky, A. A., Boal, A. K. & Barton, J. K. Direct electrochemistry of endonuclease III in the presence and absence of DNA. J. Am. Chem. Soc. 128, 12082–12083 (2006).

    Article  CAS  PubMed  Google Scholar 

  15. Arnold, A. R., Grodick, M. A. & Barton, J. K. DNA charge transport: from chemical principles to the cell. Cell Chem. Biol. 23, 183–197 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. O’Brien, E., Silva, R. M. & Barton, J. K. Redox signaling through DNA. Isr. J. Chem. 56, 705–723 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Yavin, E. et al. Protein–DNA charge transport: redox activation of a DNA repair protein by guanine radical. Proc. Natl Acad. Sci. USA 102, 3546–3551 (2005).

    Article  CAS  PubMed  Google Scholar 

  18. Boal, A. K. et al. Redox signaling between DNA repair proteins for efficient lesion detection. Proc. Natl Acad. Sci. USA 106, 15237–15242 (2009).

    Article  PubMed  Google Scholar 

  19. D’Agostino, V. G. et al. Functional analysis of MUTYH mutated proteins associated with familial adenomatous polyposis. DNA Repair 9, 700–707 (2010).

    Article  CAS  PubMed  Google Scholar 

  20. Kundu, S., Brinkmeyer, M. K., Livingston, A. L. & David, S. S. Adenine removal activity and bacterial complementation with the human MutY homologue (MUTYH) and Y165C, G382D, P391L and Q324R variants associated with colorectal cancer. DNA Repair (Amst.) 8, 1400–1410 (2009).

    Article  CAS  Google Scholar 

  21. Porello, S. L., Leyes, A. E. & David, S. S. Single-turnover and pre-steady-state kinetics of the reaction of the adenine glycosylase MutY with mismatch-containing DNA substrates. Biochemistry 37, 14756–14764 (1998).

    Article  CAS  PubMed  Google Scholar 

  22. Rich, R. L. & Myszka, D. G. Higher-throughput, label-free, real-time molecular interaction analysis. Anal. Biochem 361, 1–6 (2007).

    Article  CAS  PubMed  Google Scholar 

  23. Profrock, D. & Prange, A. Inductively coupled plasma-mass spectrometry (ICP-MS) for quantitative analysis in environmental and life sciences: a review of challenges, solutions, and trends. Appl. Spectrosc. 66, 843–868 (2012).

    Article  CAS  PubMed  Google Scholar 

  24. Pheeney, C. G., Arnold, A. R., Grodick, M. A. & Barton, J. K. Multiplexed electrochemistry of DNA-bound metalloproteins. J. Am. Chem. Soc. 135, 11869–11878 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Boon, E. M., Salas, J. E. & Barton, J. K. An electrical probe of protein–DNA interactions on DNA-modified surfaces. Nat. Biotechnol. 20, 282–286 (2002).

    Article  CAS  PubMed  Google Scholar 

  26. Kelley, S. O., Barton, J. K., Jackson, N. M. & Hill, M. G. Electrochemistry of methylene blue bound to a DNA-modified electrode. Bioconjug. Chem. 8, 31–37 (1997).

    Article  CAS  PubMed  Google Scholar 

  27. Pope, M. A. & David, S. S. DNA damage recognition and repair by the murine MutY homologue. DNA Repair 4, 91–102 (2005).

    Article  CAS  PubMed  Google Scholar 

  28. Johnson, M. K., Duderstadt, R. E. & Duin, E. C. Biological and synthetic [Fe3S4] clusters. Adv. Inorg. Chem. 47, 1–82 (1999).

    Article  CAS  Google Scholar 

  29. Sweeney, W. V. & Rabinowitz, J. C. Proteins containing 4Fe-4S clusters: an overview. Annu. Rev. Biochem 49, 139–161 (1980).

    Article  CAS  PubMed  Google Scholar 

  30. Duff, J. L. C., Breton, J. L. J., Butt, J. N., Armstrong, F. A. & Thomson, A. J. Novel redox chemistry of [3Fe-4S] clusters: Electrochemical characterization of the all-Fe(ii) form of the [3Fe-4S] cluster generated reversibly in various proteins and its spectroscopic investigation in Sulfolobus acidocaldarius ferredoxin. J. Am. Chem. Soc. 118, 8593–8603 (1996).

    Article  CAS  Google Scholar 

  31. Netz, D. J. A. et al. Eukaryotic DNA polymerases require an iron–sulfur cluster for the formation of active complexes. Nat. Chem. Biol. 8, 125–132 (2012).

    Article  CAS  Google Scholar 

  32. Good, N. E. et al. Hydrogen ion buffers for biological research. Biochemistry 5, 467 (1966).

    Article  CAS  PubMed  Google Scholar 

  33. Ugwu, S. O. The effect of buffers on protein conformational stability. Pharm. Technol. 28, 86–108 (2004).

    CAS  Google Scholar 

  34. Kundu, S., Brinkmeyer, M. K. & Eigenheer, R. A. Ser 524 is a phosphorylation site in MUTYH and Ser 524 mutations alter 8-oxoguanine (OG): a mismatch recognition. DNA Repair 9, 1026–1037 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Dorn, J., Ferrari, E., Imhof, R., Ziegler, N. & Hubscher, U. Regulation of human MutYH DNA glycosylase by the E3 ubiquitin ligase mule. J. Biol. Chem. 289, 7049–7058 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Asso, M., Guigliarelli, B., Yagi, T. & Bertrand, P. EPR and redox properties of Desulfovibrio vulgaris Miyazaki hydrogenase: comparison with the Ni–Fe enzyme from Desulfovibrio gigas. Biochim Biophys. Acta 1122, 50–56 (1992).

    Article  CAS  PubMed  Google Scholar 

  37. Kowal, A. T. et al. Effect of cysteine to serine mutations on the properties of the [4Fe-4S] center in Escherichia coli fumarate reductase. Biochemistry 34, 12284–12293 (1995).

    Article  CAS  PubMed  Google Scholar 

  38. Golinelli, M. P., Chmiel, N. H. & David, S. S. Site-directed mutagenesis of the cysteine ligands to the [4Fe-4S] cluster of Escherichia coli MutY. Biochemistry 38, 6997–7007 (1999).

    Article  CAS  PubMed  Google Scholar 

  39. Bai, H. et al. Functional characterization of two human MutY homolog (hMYH) missense mutations (R227W and V232F) that lie within the putative hMSH6 binding domain and are associated with hMYH polyposis. Nucleic Acids Res. 33, 597–604 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Ali, M. et al. Characterization of mutant MUTYH proteins associated with familial colorectal cancer. Gastroenterology 135, 499–507 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Bai, H. et al. Functional characterization of human MutY homolog (hMYH) missense mutation (R231L) that is linked with hMYH-associated polyposis. Cancer Lett. 250, 74–81 (2007).

    Article  CAS  PubMed  Google Scholar 

  42. Goto, M. et al. Adenine DNA glycosylase activity of 14 human MutY homolog (MUTYH) variant proteins found in patients with colorectal polyposis and cancer. Human Mutat. 31, E1861–1874 (2010).

    Article  CAS  Google Scholar 

  43. Fleischmann, C. et al. Comprehensive analysis of the contribution of germline MYH variation to early-onset colorectal cancer. Int. J. Cancer 109, 554–558 (2004).

    Article  CAS  PubMed  Google Scholar 

  44. Luncsford, P. J. et al. A structural hinge in eukaryotic MutY homologues mediates catalytic activity and Rad9-Rad1-Hus1 checkpoint complex interactions. J. Mol. Biol. 403, 351–370 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

The authors thank T. Huston of the W.M. Keck Lab in the Department of Earth & Environmental Sciences at the University of Michigan for ICP-HRMS analyses. This work was funded in part by a Ruth L. Kirschstein National Research Service Award (GM095065 to J.A.C.), a National Institutes of Health (NIH) grant (R35 GM118101) and an H.W. Vahlteich Professorship (to D.H.S.), a Ruth L. Kirschstein National Research Service Award and American Society of Clinical Oncology Young Investigator Award (to K.M.), grant 1R01CA197350 (to S.B.G.), a USC Norris Comprehensive Cancer Center Support Grant (CA014089 to S.B.G.), an award from the Ming Hsieh Institute for Engineering—Medicine for Cancer, and support from Daniel and Maryann Fong and the Anton B. Burg Foundation (to S.B.G.). P.L.B., E.O.B. and J.K.B. acknowledge the NIH (GM126904 to J.K.B.) and Moore Foundation for financial support. E.O.B. acknowledges NIH training grant T32-GM07616 and a Ralph Parsons Fellowship for support.

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K.M., J.C., P.B., E.O., D.S., J.B. and S.G. conceived and designed the experiments. K.M., J.C. and P.B. co-wrote the paper with input from all authors. R.S., L.R., M.M., J.O. and G.L. contributed materials and analysis tools. K.M., J.C. and P.B. performed the experiments.

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Correspondence to David H. Sherman, Jacqueline K. Barton or Stephen B. Gruber.

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McDonnell, K.J., Chemler, J.A., Bartels, P.L. et al. A human MUTYH variant linking colonic polyposis to redox degradation of the [4Fe4S]2+ cluster. Nature Chem 10, 873–880 (2018). https://doi.org/10.1038/s41557-018-0068-x

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