Skip to main content

DNA with a 2-Pyridyldithio Group at the C2' Atom: A Promising Tool for the Crosslinking of the MutS Protein Preserving Its Functional Activity

Abstract

Noncovalent interactions between DNA and proteins are vital for cells. DNA-protein crosslinking may occur due to various endogenous and exogenous factors, as well as chemotherapeutic agents, and often affects the performance of DNA-binding proteins. Oligodeoxyribonucleotides with a reactive group can serve as a tool to investigate the structure and action of DNA-binding proteins. Here, we report the crosslinking of MutS, which is a sensor protein of DNA mismatch repair (MMR) in Escherichia coli, via the protein’s cysteine. This was realized via DNA that harboured a 2′-deoxy-2′-[3-(2-pyridyldithio)propionamide] group as part of a nucleoside at a given position, and two fluorophores. The proposed DNA duplex consisted of complementary oligonucleotides with breaks in the top and bottom strands. This feature allowed us to introduce the reactive group at different positions and made it possible to synthesize only the central fragment of the DNA with a minimal length. The MutS-DNA conjugate was obtained by thiol-disulfide exchange with quantitative yield and separated from the unreacted DNA by size-exclusion chromatography. Fluorescence resonance energy transfer was used to show that MutS remained functionally active within the conjugate, since the protein was able to change its conformation and DNA conformation as well.

This is a preview of subscription content, access via your institution.

Fig. 1.
Fig. 2.
Fig. 3.
Fig. 4.
Fig. 5.
Fig. 6.
Fig. 7.
Fig. 8.
Fig. 9.

REFERENCES

  1. Dolinnaya, N., Zubin, E., Kubareva, E., Zatsepin, T., and Oretskaya, T., Curr. Org. Chem., 2009, vol. 13, pp. 1029–1049. https://doi.org/10.2174/138527209788680745

    CAS  Article  Google Scholar 

  2. Ilina, E.S., Khodyreva, S.N., and Lavrik, O.I., Biochimie, 2018, vol. 150, pp. 88–99. https://doi.org/10.1016/j.biochi.2018.04.027

    CAS  Article  PubMed  Google Scholar 

  3. Ide, H., Nakano, T., Salem, A.M.H., and Shoulkamy, M.I., DNA Repair, 2018, vol. 71, pp. 190–197. https://doi.org/10.1016/j.dnarep.2018.08.024

    CAS  Article  PubMed  Google Scholar 

  4. Stasińska, A.R., Putaj, P., and Chmielewski, M.K., Bioorg. Chem., 2020, vol. 95, p. 103518. https://doi.org/10.1016/j.bioorg.2019.103518

    CAS  Article  PubMed  Google Scholar 

  5. Wong, S.S. and Jameson, D.M., Chemistry of Protein and Nucleic Acid Cross-Linking and Conjugation, 2nd ed., Boca Raton, FL: CRC, 2011.

    Book  Google Scholar 

  6. Khodyreva, S. and Lavrik, O., DNA Repair (Amst.), 2020, vol. 90, p. 102847. https://doi.org/10.1016/j.dnarep.2020.102847

    CAS  Article  Google Scholar 

  7. Hyjek-Składanowska, M., Stasinska, A.R., Napiórkowska-Gromadzka, A., Bartłomiejczak, A., Seth, P.P., Chmielewski, M.K., and Nowotny, M., Bioorg. Med. Chem., 2020. https://doi.org/10.1016/j.bmc.2020.115741

  8. Friedhoff, P., Manelyte, L., Giron-Monzon, L., Winkler, I., Groothuizen, F.S., and Sixma, T.K., Methods Enzymol., 2017, vol. 592, pp. 77–101. https://doi.org/10.1016/bs.mie.2017.03.025

    CAS  Article  PubMed  Google Scholar 

  9. Monakhova, M., Ryazanova, A., Hentschel, A., Viryasov, M., Oretskaya, T., Friedhoff, P., and Kubareva, E., J. Chromatogr. A, 2015, vol. 1389, pp. 19–27. https://doi.org/10.1016/j.chroma.2015.02.045

    CAS  Article  PubMed  Google Scholar 

  10. Monakhova, M., Ryazanova, A., Kunetsky, V., Li, P., Shilkin, E., Kisil, O., Rao, D.N., Oretskaya, T., Friedhoff, P., and Kubareva, E., Biochimie, 2020, vols. 171–172, pp. 43–54. https://doi.org/10.1016/j.biochi.2020.02.004

    CAS  Article  PubMed  Google Scholar 

  11. Metelev, V.G., Kubareva, E.A., Vorob’eva, O.V., Romanenkov, A.S., and Oretskaya, T.S., FEBS Lett., 2003, vol. 538, pp. 48–52. https://doi.org/10.1016/S0014-5793(03)00122-4

    CAS  Article  PubMed  Google Scholar 

  12. Vorob’eva, O.V, Romanenkov, A.S., Metelev, V.G., Kariagina, A.S., Lavrova, N.V., Oretskaia, T.S., and Kubareva, E.A., Mol. Biol. (Moscow), 2003, vol. 37, pp. 906–915.

    Google Scholar 

  13. Romanenkov, A.S., Kisil, O.V., Zatsepin, T.S., Yamskova, O.V., Karyagina, A.S., Metelev, V.G., Oretskaya, T.S., and Kubareva, E.A., Biochemistry (Moscow), 2006, vol. 71, pp. 1341–1349. https://doi.org/10.1134/s0006297906120091

    CAS  Article  PubMed  Google Scholar 

  14. Abrosimova, L.A., Samsonova, A.R., Perevyazova, T.A., Yunusova, A.K., Artyukh, R.I., Romanova, E.A., Zheleznaya, L.A., Oretskaya, T.S., and Kubareva, E.A., Mol. Biol. (Moscow.), 2020, vol. 54, pp. 599–610. https://doi.org/10.1134/S0026893320040020

  15. Metelev, V., Romanenkov, A., Kubareva, E., Zubin, E., Polouchine, N., Zatsepin, T., Molochkov, N., and Oretskaya, T., IUBMB Life, 2006, vol. 58, pp. 654–658. https://doi.org/10.1080/15216540600981750

    CAS  Article  PubMed  Google Scholar 

  16. Heinze, R.J., Sekerina, S., Winkler, I., Biertümpfel, C., Oretskaya, T.S., Kubareva, E., and Friedhoff, P., Mol. Biosyst., 2012, vol. 8, pp. 1861–1864. https://doi.org/10.1039/c2mb25086a

    CAS  Article  PubMed  Google Scholar 

  17. Cristóvão, M., Sisamakis, E., Hingorani, M.M., Marx, A.D., Jung, C.P., Rothwell, P.J., Seidel, C.A.M., and Friedhoff, P., Nucleic Acids Res., 2012, vol. 40, pp. 5448–5464. https://doi.org/10.1093/nar/gks138

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  18. Warren, J.J., Pohlhaus, T.J., Changela, A., Iyer, R.R., Modrich, P.L., and Beese, L.S., Mol. Cell, 2007, vol. 26, pp. 579–592. https://doi.org/10.1016/j.molcel.2007.04.018

    CAS  Article  PubMed  Google Scholar 

  19. Biswas, I. and Hsieh, P., J. Biol. Chem., 1997, vol. 272, pp. 13355–13364. https://doi.org/10.1074/jbc.272.20.13355

    CAS  Article  PubMed  Google Scholar 

  20. Natrajan, G., Nucleic Acids Res., 2003, vol. 31, pp. 4814–4821. https://doi.org/10.1093/nar/gkg677

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  21. Perevoztchikova, S.A., Romanova, E.A., Oretskaya, T.S., Friedhoff, P., and Kubareva, E.A., Acta Naturae, 2013, vol. 5, pp. 17–34. https://doi.org/10.32607/20758251-2013-5-3-17-34

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  22. Putnam, C.D., Proc. Natl. Acad. Sci. U. S. A., 2020, vol. 117, pp. 20351–20353. https://doi.org/10.1073/pnas.2013560117

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  23. Bhairosing-Kok, D., Groothuizen, F.S., Fish, A., Dharadhar, S., Winterwerp, H.H.K., and Sixma, T.K., Nucleic Acids Res., 2019, vol. 47, pp. 8888–8898. https://doi.org/10.1093/nar/gkz649

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  24. Liu, J., Hanne, J., Britton, B.M., Bennett, J., Kim, D., Lee, J.-B., and Fishel, R., Nature, 2016, vol. 539, pp. 583–587. https://doi.org/10.1038/nature20562

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  25. Perevozchikova, S.A., Trikin, R.M., Heinze, R.J., Romanova, E.A., Oretskaya, T.S., Friedhoff, P., and Kubareva, E.A., PLoS One, 2014, vol. 9. e104963. https://doi.org/10.1371/journal.pone.0104963

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  26. Hura, G.L., Tsai, C.-L., Claridge, S.A., Mendillo, M.L., Smith, J.M., Williams, G.J., Mastroianni, A.J., Alivisatos, A.P., Putnam, C.D., Kolodner, R.D., and Tainer, J.A., Proc. Natl. Acad. Sci. U. S. A., 2013, vol. 110, pp. 17308–17313. https://doi.org/10.1073/pnas.1308595110

    Article  PubMed  PubMed Central  Google Scholar 

  27. Heinze, R.J., Giron-Monzon, L., Solovyova, A., Elliot, S.L., Geisler, S., Cupples, C.G., Connolly, B.A., and Friedhoff, P., Nucleic Acids Res., 2009, vol. 37, pp. 4453–4463. https://doi.org/10.1093/nar/gkp380

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  28. Kramer, B., Kramer, W., and Fritz, H.-J., Cell, 1984, vol. 38, pp. 879–887. https://doi.org/10.1016/0092-8674(84)90283-6

    CAS  Article  PubMed  Google Scholar 

  29. Lamers, M.H., Georgijevic, D., Lebbink, J.H., Winterwerp, H.H.K., Agianian, B., de Wind, N., and Sixma, T.K., J. Biol. Chem., 2004, vol. 279, pp. 43879–43885. https://doi.org/10.1074/jbc.M406380200

    CAS  Article  PubMed  Google Scholar 

  30. Lebbink, J.H.G., Georgijevic, D., Natrajan, G., Fish, A., Winterwerp, H.H.K., Sixma, T.K., and de Wind, N., EMBO J., 2006, vol. 25, pp. 409–419. https://doi.org/10.1038/sj.emboj.7600936

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  31. Lamers, M.H., Perrakis, A., Enzlin, J.H., Winterwerp, H.H.K., de Wind, N., and Sixma, T.K., Nature, 2000, vol. 407, pp. 711–717. https://doi.org/10.1038/35037523

    CAS  Article  PubMed  Google Scholar 

  32. Groothuizen, F.S., Fish, A., Petoukhov, M.V., Reumer, A., Manelyte, L., Winterwerp, H.H.K., Marinus, M.G., Lebbink, J.H.G., Svergun, D.I., Friedhoff, P., and Sixma, T.K., Nucleic Acids Res., 2013, vol. 41, pp. 8166–8181. https://doi.org/10.1093/nar/gkt582

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  33. Bjornson, K.P. and Modrich, P., J. Biol. Chem., 2003, vol. 278, pp. 18557–18562. https://doi.org/10.1074/jbc.M301101200

    CAS  Article  PubMed  Google Scholar 

  34. Manelyte, L., Urbanke, C., Giron-Monzon, L., and Friedhoff, P., Nucleic Acids Res., 2006, vol. 34, pp. 5270–5279. https://doi.org/10.1093/nar/gkl489

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  35. Feng, G. and Winkler, M.E., BioTechniques, 1995, vol. 19, pp. 956–965.

    CAS  PubMed  Google Scholar 

  36. Lebbink, J.H.G., Fish, A., Reumer, A., Natrajan, G., Winterwerp, H.H.K., and Sixma, T.K., J. Biol. Chem., 2010, vol. 285, pp. 13131–13141. https://doi.org/10.1074/jbc.M109.066001

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  37. Ede, N.J., Tregear, G.W., and Haralambidis, J., Bioconjug. Chem., 1994, vol. 5, pp. 373–378. https://doi.org/10.1021/bc00028a016

    CAS  Article  PubMed  Google Scholar 

  38. Qiu, R., Sakato, M., Sacho, E.J., Wilkins, H., Zhang, X., Modrich, P., Hingorani, M.M., Erie, D.A., and Weninger, K.R., Proc. Natl. Acad. Sci. U. S. A., 2015, vol. 112, pp. 10914–10919. https://doi.org/10.1073/pnas.1505655112

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  39. LeBlanc, S.J., Gauer, J.W., Hao, P., Case, B.C., Hingorani, M.M., Weninger, K.R., and Erie, D.A., Nucleic Acids Res., 2018, vol. 46, pp. 10782–10795. https://doi.org/10.1093/nar/gky865

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  40. Groothuizen, F.S., Winkler, I., Cristóvão, M., Fish, A., Winterwerp, H.H., Reumer, A., Marx, A.D., Hermans, N., Nicholls, R.A., Murshudov, G.N., Lebbink, J.H., Friedhoff, P., and Sixma, T.K., Elife, 2015, vol. 4. https://doi.org/10.7554/eLife.06744

  41. Hao, P., LeBlanc, S.J., Case, B.C., Elston, T.C., Hingorani, M.M., Erie, D.A., and Weninger, K.R., Proc. Natl. Acad. Sci. U. S. A., 2020, vol. 117, pp. 17775–17784. https://doi.org/10.1073/pnas.1918517117

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  42. Liu, J., Lee, R., Britton, B.M., London, J.A., Yang, K., Hanne, J., Lee, J.-B., and Fishel, R., Nat. Commun., 2019, vol. 10, p. 5294. https://doi.org/10.1038/s41467-019-13191-5

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  43. Herráez, A., Biochem. Mol. Biol. Educ., 2006, vol. 34, pp. 255–261. https://doi.org/10.1002/bmb.2006.494034042644

    Article  PubMed  Google Scholar 

Download references

ACKNOWLEDGMENTS

The authors are grateful to Dr. R. Heinz (Institute for Biochemistry, Justus Liebig University, Giessen, Germany) for invaluable help in carrying out experiments on crosslinking.

Funding

The study was supported by the DFG (project no. GRK 1384) and RFBR-DFG (project no. 14-04-91343) within the framework of the educational program “International Research Training Groups with the Participation of Young Scientists.”

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to M. V. Monakhova.

Ethics declarations

COMPLIANCE WITH ETHICAL STANDARDS

This article does not describe any research involving humans and animals.

CONFLICT OF INTERESTS

The authors declare they have no conflicts of interest.

Additional information

Translated by D. Martynova

Abbreviations: FRET, fluorescent resonant energy transfer; MANT-ADP,  2′/3′-O-(N-methylanthraniloyl)adenosine-5′-diphosphate; MMR, DNA mismatch repair system; PDB, Protein Data Bank database; U, 2′-deoxy-2′-[3-(2-pyridyldithio)propionamido]uridine residue. The prefix “d” (deoxy) has been omitted for oligodeoxyribonucleotides and DNA duplexes.

Corresponding author: phone: +7 (495) 939-31-48.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Perry, S.A., Kubareva, E.A., Monakhova, M.V. et al. DNA with a 2-Pyridyldithio Group at the C2' Atom: A Promising Tool for the Crosslinking of the MutS Protein Preserving Its Functional Activity. Russ J Bioorg Chem 47, 447–460 (2021). https://doi.org/10.1134/S1068162021020205

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1134/S1068162021020205

Keywords:

  • reactive DNA
  • DNA-protein crosslinking
  • thiol-disulfide exchange
  • DNA mismatch repair system (MMR)
  • MutS