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Unnatural substrates reveal the importance of 8-oxoguanine for in vivo mismatch repair by MutY

Nature Chemical Biology volume 4, pages 51–58 (2008)Cite this article

Abstract

Escherichia coli MutY has an important role in preventing mutations associated with the oxidative lesion 7,8-dihydro-8-oxo-2′-deoxyguanosine (OG) in DNA by excising adenines from OG·A mismatches as the first step of base excision repair. To determine the importance of specific steps in the base pair recognition and base removal process of MutY, we have evaluated the effects of modifications of the OG·A substrate on the kinetics of base removal, mismatch affinity and repair to G-C in an E. coli–based assay. Notably, adenine modification was tolerated in the cellular assay, whereas modification of OG resulted in minimal cellular repair. High affinity for the mismatch and efficient base removal required the presence of OG. Taken together, these results suggest that the presence of OG is a critical feature that is necessary for MutY to locate OG·A mismatches and select the appropriate adenines for excision to initiate repair in vivo before replication.

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Figure 1: Recognition of an OG·A mismatch by D144N BsMY and modifications made to probe features of mismatch repair.
Figure 2: Kinetic analysis of the glycosylase activity of MutY.
Figure 3: Cell-based MutY-mediated repair assay.

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References

  1. Iyer, R.R., Pluciennik, A., Burdett, V. & Modrich, P.L. DNA mismatch repair: functions and mechanisms. Chem. Rev. 106, 302–323 (2006).

    Article  CAS  Google Scholar 

  2. Su, S.-S., Lahue, R.S., Au, K.G. & Modrich, P. Mispair specificity of methyl-directed DNA mismatch correction. J. Biol. Chem. 263, 6829–6835 (1988).

    CAS  PubMed  Google Scholar 

  3. Lu, A.-L. & Chang, D.-Y. Repair of single base-pair transversion mismatches of Escherichia coli in vitro: correction of certain A/G mismatches is independent of dam methylation and host mut HLS gene functions. Genetics 118, 593–600 (1988).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Radicella, J.P., Clark, E.A. & Fox, M.S. Some mismatch repair activities in Escherichia coli. Proc. Natl. Acad. Sci. USA 85, 9674–9678 (1988).

    Article  CAS  Google Scholar 

  5. Nghiem, Y., Cabrera, M., Cupples, C.G. & Miller, J.H. The mutY gene: A mutator locus in Escherichia coli that generates G:C to T:A transversions. Proc. Natl. Acad. Sci. USA 85, 2709–2713 (1988).

    Article  CAS  Google Scholar 

  6. Au, K.G., Cabrera, M., Miller, J.H. & Modrich, P. Escherichia coli mutY gene product is required for specific AG to CG mismatch correction. Proc. Natl. Acad. Sci. USA 85, 9163–9166 (1988).

    Article  CAS  Google Scholar 

  7. Au, K.G., Clark, S., Miller, J.H. & Modrich, P. Escherichia coli mutY gene encodes an adenine glycosylase active on G-A mispairs. Proc. Natl. Acad. Sci. USA 86, 8877–8881 (1989).

    Article  CAS  Google Scholar 

  8. Lu, A.-L. & Chang, D.-Y. A novel nucleotide excision repair for the conversion of an A/G mismatch to C/G base pair in E. coli. Cell 54, 805–812 (1988).

    Article  CAS  Google Scholar 

  9. David, S.S. & Williams, S.D. Chemistry of glycosylases and endonucleases involved in base-excision repair. Chem. Rev. 98, 1221–1261 (1998).

    Article  CAS  Google Scholar 

  10. David, S.S., O'Shea, V.L. & Kundu, S. Base-excision repair of oxidative DNA damage. Nature 447, 941–950 (2007).

    Article  CAS  Google Scholar 

  11. Sung, J.-S. & Demple, B. Roles of base excision repair subpathways in correcting oxidized abasic sites in DNA. FEBS J. 273, 1620–1629 (2006).

    Article  CAS  Google Scholar 

  12. Michaels, M.L. & Miller, J.H. The GO system protects organisms from the mutagenic effect of the spontaneous lesion 8-hydroxyguanine (7,8-dihydro-8-oxoguanine). J. Bacteriol. 174, 6321–6325 (1992).

    Article  CAS  Google Scholar 

  13. Michaels, M.L., Tchou, J., Grollman, A.P. & Miller, J.H. A repair system for 8-oxo-7,8-dihydrodeoxyguanosine. Biochemistry 31, 10964–10968 (1992).

    Article  CAS  Google Scholar 

  14. Michaels, M.L., Cruz, C., Grollman, A.P. & Miller, J.H. Evidence that MutY and MutM combine to prevent mutations by an oxidatively damaged form of guanine in DNA. Proc. Natl. Acad. Sci. USA 89, 7022–7025 (1992).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  16. Sampson, J.R., Jones, S., Dolwani, S. & Cheadle, J.P. MutYH (MYH) and colorectal cancer. Biochem. Soc. Trans. 33, 679–683 (2005).

    Article  CAS  Google Scholar 

  17. 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  Google Scholar 

  18. Francis, A.W., Helquist, S.A., Kool, E.T. & David, S.S. Probing the requirements for recognition and catalysis in Fpg and MutY with nonpolar adenine isosteres. J. Am. Chem. Soc. 125, 16235–16242 (2003).

    Article  CAS  Google Scholar 

  19. Chmiel, N.H., Golinelli, M.-P., Francis, A.W. & David, S.S. Efficient recognition of substrates and substrate analogs by the adenine glycosylase MutY requires the C-terminal domain. Nucleic Acids Res. 29, 553–564 (2001).

    Article  CAS  Google Scholar 

  20. McCann, J.A.B. & Berti, P.J. Adenine release is fast in MutY-catalyzed hydrolysis of G:A and 8-oxo-G:A DNA mismatches. J. Biol. Chem. 278, 29587–29592 (2003).

    Article  CAS  Google Scholar 

  21. Pope, M.A., Porello, S.L. & David, S.S. E. coli AP endonucleases enhance the turnover of the adenine glycosylase MutY with G:A substrates. J. Biol. Chem. 277, 22605–22615 (2002).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  23. Hang, B. & Singer, B. Protein-Protein interactions involving DNA glycosylases. Chem. Res. Toxicol. 16, 1181–1195 (2003).

    Article  CAS  Google Scholar 

  24. Fromme, J.C., Banerjee, A., Huang, S.J. & Verdine, G.L. Structural basis for removal of adenine mispaired with 8-oxoguanine by MutY adenine DNA glycosylase. Nature 427, 652–656 (2004).

    Article  CAS  Google Scholar 

  25. Chmiel, N.H., Livingston, A.L. & David, S.S. Insight into the functional consequences of inherited variants of the hMYH adenine glycosylase associated with colorectal cancer: complementation assays with hMYH variants and pre-steady-state kinetics of the corresponding mutated E. coli enzymes. J. Mol. Biol. 327, 431–443 (2003).

    Article  CAS  Google Scholar 

  26. Pearson, C.G., Shikazono, N., Thacker, J. & O'Neill, P.O. Enhanced mutagenic potential of 8-oxo-7,8-dihydroguanine when present within a cluster DNA damage site. Nucleic Acids Res. 32, 263–270 (2004).

    Article  CAS  Google Scholar 

  27. Shikazono, N., Pearson, C., O'Neill, P.O. & Thacker, J. The roles of specific glycosylases in determining the mutagenic consequences of clustered DNA base damage. Nucleic Acids Res. 34, 3722–3730 (2006).

    Article  CAS  Google Scholar 

  28. Zhao, J. & Winkler, M.E. Reduction of the GC to TA transversion mutation by overexpression of MutS in E. coli K-12. J. Bacteriol. 182, 5025–5028 (2000).

    Article  CAS  Google Scholar 

  29. Bai, H. & Lu, A.-L. Physical and functional interactions between Escherichia coli MutY glycosylase and mismatch repair protein MutS. J. Bacteriol. 189, 902–910 (2007).

    Article  CAS  Google Scholar 

  30. Berti, P.J. & McCann, J.A.B. Toward a detailed understanding of base excision repair enzymes: transition state and mechanistic analyses of N-glycoside hydrolysis and N-glycoside transfer. Chem. Rev. 106, 506–555 (2006).

    Article  CAS  Google Scholar 

  31. Chepanoske, C.L., Porello, S.P., Fujiwara, T., Sugiyama, H. & David, S.S. Investigation of substrate recognition by E. coli MutY using substrate analogs. Nucleic Acids Res. 27, 3197–3204 (1999).

    Article  CAS  Google Scholar 

  32. 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  Google Scholar 

  33. 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  Google Scholar 

  34. Miller, J.H. A Short Course in Bacterial Genetics 193–211 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA, 1992).

    Google Scholar 

  35. Wehrli, W., Knusel, F., Schmid, K. & Staehlin, M. Interaction of rifamycin with bacterial RNA polymerase. Proc. Natl. Acad. Sci. USA 61, 667–673 (1968).

    Article  CAS  Google Scholar 

  36. Livingston, A.L., Kundu, S., Henderson-Pozzi, M., Anderson, D.W. & David, S.S. Insight into the roles of tyrosine 82 and glycine 253 in the Escherichia coli adenine glycosylase MutY. Biochemistry 44, 14179–14190 (2005).

    Article  CAS  Google Scholar 

  37. Record, M.T., Ha, J.-H. & Fisher, M.A. Analysis of equilibrium and kinetic measurements to determine thermodynamic origins of stability and specificity and mechanism of formation of site-specific complexes between proteins and helical DNA. Methods Enzymol. 208, 291–343 (1991).

    Article  CAS  Google Scholar 

  38. Bernards, A.S., Miller, J.K., Bao, K.K. & Wong, I. Flipping duplex DNA inside out: a double base-flipping reaction mechanism by Escherichia coli MutY adenine glycosylase. J. Biol. Chem. 277, 20960–20964 (2002).

    Article  CAS  Google Scholar 

  39. Porello, S.L., Williams, S.D., Kuhn, H., Michaels, M.L. & David, S.S. Specific recognition of substrate analogs by the DNA mismatch repair enzyme MutY. J. Am. Chem. Soc. 118, 10684–10692 (1996).

    Article  CAS  Google Scholar 

  40. Pope, M.A., Chmiel, N.H. & David, S.S. Insight into the functional consequences of hMYH variants associated with colorectal cancer: distinct differences in the adenine glycosylase activity and the response to AP endonuclease of Y150C and G365D murine MYH. DNA Repair (Amst.) 4, 315–325 (2005).

    Article  CAS  Google Scholar 

  41. Maki, A.S., Kim, T. & Kool, E.T. Direct comparisons of A-strand and T-strand minor groove interactions in DNA curvature at A tracts. Biochemistry 43, 1102–1110 (2004).

    Article  CAS  Google Scholar 

  42. Sambrook, J. & Maniatis, T. Molecular Cloning: a Laboratory Manual, 1.33–1.75 (Cold Spring Harbor Press, Cold Spring Harbor, New York, USA, 1989).

    Google Scholar 

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Acknowledgements

We thank B. Wilcock, S. Kundu and M.A. Pope for technical assistance. We also thank M. Marinus (University of Massachusetts) for providing the AB1157, GM7724 (mutY::Cam) and KM75 (mutS465::Tet) E. coli strains. J. Miller and M. Michaels (University of California, Los Angeles) provided the pKKYEco plasmid and JM101 (mutY−) E. coli strain. This work was supported by US National Institutes of Health (NIH) grants to S.S.D. (CA67985) and E.T.K. (GM072705), and by NIH predoctoral traineeships to A.L.L. (GM08537) and V.L.O. (GM08537 and CA093247). The DNA sequencing facility at the University of Utah Medical School is supported in part by an NIH National Cancer Institute grant (5P30CA43014).

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Author notes

  1. Alison L Livingston and Valerie L O'Shea: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Chemistry, University of Utah, 315 South 1400 East, Salt Lake City, 84112, Utah, USA

    Alison L Livingston & Valerie L O'Shea

  2. Department of Chemistry, Stanford University, Stauffer 1, Stanford, 94305, California, USA

    Taewoo Kim & Eric T Kool

  3. Department of Chemistry, University of California, Davis, One Shields Avenue, Davis, 95616, California, USA

    Sheila S David

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  1. Alison L Livingston
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Contributions

A.L.L., V.L.O. and S.S.D. designed the experiments. A.L.L. and V.L.O. performed the experiments. T.K. synthesized the Z3-containing duplex 2 oligonucleotide. S.S.D. and E.T.K. supervised all experiments. A.L.L., V.L.O. and S.S.D. wrote the manuscript. E.T.K. read and made comments on the manuscript.

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Correspondence to Sheila S David.

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Livingston, A., O'Shea, V., Kim, T. et al. Unnatural substrates reveal the importance of 8-oxoguanine for in vivo mismatch repair by MutY. Nat Chem Biol 4, 51–58 (2008). https://doi.org/10.1038/nchembio.2007.40

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