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The Role of Cysteine Residues in the Interaction of Nicking Endonuclease BspD6I with DNA

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Abstract

Nicking endonucleases (NEs) are a small, poorly studied family of restriction endonucleases. The enzymes recognize a target sequence in DNA, but catalyze the hydrolysis of only one strand. The mechanism of their action is important to study because NEs with new specificities are necessary to design to solve the practical tasks of biotechnology. One of the modern approaches for investigation of protein-nucleic acid interactions is fluorescence spectroscopy, which involves the introduction of fluorophores into proteins, mainly through Cys residues due to the high reactivity of their thiol group. To implement this approach, it is necessary to clarify the role of Cys residues in the functioning of the native protein and the possible consequences of their modification. Crosslinking was used to study whether Cys residues are close to DNA in the complex with NE BspD6I. Reactions were carried out using the wild-type enzyme, its mutant form NE BspD6I(C11S/C160S), and modified DNA duplexes containing the 2-pyridyldisulfide group at the C2' atom of the sugar-phosphate moiety in different positions of the oligonucleotide strand. The Cys residues of NE BspD6I were for the first time shown to be in close proximity to DNA during the binding process, including the step of a nonspecific complex formation. The substitutions C11S and C160S in the N-terminal domain of the enzyme slightly decreased the efficiency of substrate hydrolysis. Construction of a cysteine-free NE BspD6I variant and examination of its properties will provide additional information about the functional significance of the Cys residues for this unique enzyme.

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REFERENCES

  1. Abrosimova L.A., Kisil O.V., Romanova E.A., Oretskaya T.S., Kubareva E.A. 2019. Nicking endonucleases as unique tools for biotechnology and gene engineering. Russ. J. Bioorg. Chem. 45 (5), 303–320.

    Article  CAS  Google Scholar 

  2. Gabsalilow L., Schierling B., Friedhoff P., Pingoud A., Wende W. 2013. Site-and strand-specific nicking of DNA by fusion proteins derived from MutH and I-SceI or TALE repeats. Nucleic Acids Res.41, e83.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Van Nierop G.P., de Vries A.A.F., Holkers M., Vrijsen K.R., Gonçalves M.A. 2009. Stimulation of homology-directed gene targeting at an endogenous human locus by a nicking endonuclease. Nucleic Acids Res.37, 5725–5736.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Zheleznaya L.A., Perevyazova T.A., Alzhanova D.V., Matvienko N.I. 2001. Site-specific nicjase from Bacillus species strain D6. Biochemistry (Moscow). 66 (9), 989–993.

    CAS  PubMed  Google Scholar 

  5. Yunusova A.K., Rogulin E.A., Artyukh R.I., Zheleznaya L.A., Matvienko N.I. 2006. Nickase and a protein encoded by an open reading frame downstream from the nickase BspD6I gene form a restriction endonuclease complex. Biochemistry (Moscow). 71 (7), 815–820.

    CAS  PubMed  Google Scholar 

  6. Abrosimova L.A., Kubareva E.A., Migur A.Y., Gavshina A.V., Ryazanova A.Y., Norkin M.V., Perevyazova T.A., Wende W., Hianik T., Zheleznaya L.A., Oretskaya T.S. 2016. Peculiarities of the interaction of the restriction endonuclease BspD6I with DNA containing its recognition site. Biochim. Biophys. Acta—Proteins Proteom. 1864, 1072–1082.

    Article  CAS  Google Scholar 

  7. Kachalova G.S., Rogulin E.A., Artyukh R.I., Perevyazova T.A., Zheleznaya L.A., Matvienko N.I., Bartunik H.D. 2005. Crystallization and preliminary crystallographic analysis of the site-specific DNA nickase Nb.BspD6I. Acta Crystallogr. F: Struct. Biol. Cryst. Commun.61, 332–334.

    Article  CAS  Google Scholar 

  8. Sekerina S.A., Grishin A.V., Ryazanova A.Yu., Artyukh R.I., Rogulin E.A., Yunusova A.K., Oretskaya T.S., Zheleznaya L.A., Kubareva E.A. 2012. Oligomerization of site-specific nicking endonuclease BspD6I at high protein concentrations. Russ. J. Bioorg. Chem. 38 (4), 376–382.

    Article  CAS  Google Scholar 

  9. Abrosimova L.A., Migur A.Yu., Kubareva E.A., Vende V., Zheleznaya L.A., Oretskaya T.S. 2015. Development of inhibitors for nicking endonuclease BspD6I based on synthetic DNA fragments. Izv. Vyssh. Uchebn. Zaved.,Prikl. Khim. Biotekhnol.2, 48–59.

    Google Scholar 

  10. Abrosimova L.A., Migur A.Y., Kubareva E.A., Zatsepin T.S., Gavshina A.V., Yunusova A.K., Perevyazova T.A., Pingoud A., Oretskaya T.S. 2018. A study on endonuclease BspD6I and its stimulus-responsive switching by modified oligonucleotides. PLoS One. 13, e0207302.

    Article  PubMed  PubMed Central  Google Scholar 

  11. Yunusova A.K., Artyukh R.I., Perevyazova T.A., Abrosimova L.A., Kachalova G.S., Zheleznaya L.A. 2017. Restriction endonuclease R.BspD6I is active only in the presence of catalytically active large subunit. MEDLINE.RU. 18, 200–208.

  12. Kachalova G.S., Rogulin E.A., Yunusova A.K., Artyukh R.I., Perevyazova T.A., Matvienko N.I., Zheleznaya L.A., Bartunik H.D. 2008. Structural analysis of the heterodimeric type IIS restriction endonuclease R.BspD6I acting as a complex between a monomeric site-specific nickase and a catalytic subunit. J. Mol. Biol.384, 489–502.

    Article  CAS  PubMed  Google Scholar 

  13. Verdine G.L., Norman D.P.G. 2003. Covalent trapping of protein–DNA complexes. Annu. Rev. Biochem.72, 337–366.

    Article  CAS  PubMed  Google Scholar 

  14. Metelev V.G., Kubareva E.A., Vorob’eva O.V., Romanenkov A.S., Oretskaya T.S. 2003. Specific conjugation of DNA binding proteins to DNA templates through thiol-disulfide exchange. FEBS Lett. 538, 48–52.

    Article  CAS  PubMed  Google Scholar 

  15. Vorob’eva O.V., Romanenkov A.S., Metelev V.G., Karyagina A.S., Lavrova N.V., Oretskaya T.S., Kubareva E.A. 2003. Crosslinking of Cys142 of methyltransferase SsoII with DNA duplexes containing a single internucleotide phosphoryldisulfide link. Mol. Biol. (Moscow). 37 (5), 772–779.

    Article  Google Scholar 

  16. Metelev V., Romanenkov A., Kubareva E., Zubin E., Polouchine N., Zatsepin T., Molochkov N., Oretskaya T. 2006. Structure-based cross-linking of NF-κB p50 homodimer and decoy bearing a novel 2′-disulfide trapping site. IUBMB Life.58, 654–658.

    Article  CAS  PubMed  Google Scholar 

  17. Romanenkov A.S., Kisil O.V., Zatsepin T.S., Yamskova O.V., Karyagina A.S., Metelev V.G., Oretskaya T.S., Kubareva E.A. 2006. DNA-methyltransferase SsoII as a bifunctional protein: Features of the Interaction with the promoter region of SsoII restriction–modification genes. Biochemistry (Moscow). 71 (12), 1341–1349.

    CAS  PubMed  Google Scholar 

  18. Heinze R.J., Sekerina S., Winkler I., Biertümpfel C., Oretskaya T.S., Kubareva E., Friedhoff P. 2012. Covalently trapping MutS on DNA to study DNA mismatch recognition and signaling. Mol. Biosyst.8, 1861–1864.

    Article  CAS  PubMed  Google Scholar 

  19. Monakhova M., Ryazanova A., Hentschel A., Viryasov M., Oretskaya T., Friedhoff P., Kubareva E. 2015. Chromatographic isolation of the functionally active MutS protein covalently linked to deoxyribonucleic acid. J. Chromatogr. A.1389, 19–27.

    Article  CAS  PubMed  Google Scholar 

  20. Monakhova M., Ryazanova A., Kunetsky V., Li P., Shilkin E., Kisil O., Rao D.N., Oretskaya T., Friedhoff P., Kubareva E. 2020. Probing the DNA-binding center of the MutL protein from the Escherichia coli mismatch repair system via crosslinking and Förster resonance energy transfer. Biochimie. 171, 43–54.

    Article  PubMed  Google Scholar 

  21. Stasińska A.R., Putaj P., Chmielewski M.K. 2020. Disulfide bridge as a linker in nucleic acids’ bioconjugation: 2. A summary of practical applications. Bioorg. Chem.95, 103518.

    Article  PubMed  Google Scholar 

  22. Stasińska A.R., Putaj P., Chmielewski M.K. 2019. Disulfide bridge as a linker in nucleic acids’ bioconjugation: 1. An overview of synthetic strategies. Bioorg. Chem.92, 103223.

    Article  PubMed  Google Scholar 

  23. Leichert L.I., Jakob U. 2006. Global methods to monitor the thiol-disulfide state of proteins in vivo.Antioxid. Redox Signal. 8, 763–772.

    Article  CAS  PubMed  Google Scholar 

  24. Holliday G.L., Mitchell J.B., Thornton J.M. 2009. Understanding the functional roles of amino acid residues in enzyme catalysis. J. Mol. Biol.390, 560–577.

    Article  CAS  PubMed  Google Scholar 

  25. Luscombe N.M., Laskowski R.A., Thornton J.M. 2001. Amino acid-base interactions: A three-dimensional analysis of protein-DNA interactions at atomic level. Nucleic Acids Res.29, 2860–2874.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Luscombe N.M., Thornton J.M. 2002. Protein–DNA interactions: amino acid conservation and the effects of mutations on binding specificity. J. Mol. Biol.320, 991–1009.

    Article  CAS  PubMed  Google Scholar 

  27. Ghosh G., Duyne G.V., Ghosh S., Sigler P.B. 1995. Structure of NF-κB p50 homodimer bound to a κB site. Nature.373, 303–310.

    Article  CAS  PubMed  Google Scholar 

  28. Chen Y.-Q., Ghosh S., Ghosh G. 1998. A novel DNA recognition mode by NF-κB p65 homodimer. Nat. Struct. Biol.5, 67–73.

    Article  PubMed  Google Scholar 

  29. Escalante C.R., Shen L., Thana D., Aggaraiwal A.K. 2002. Structure of the NF-κB p50/p65 heterodimer bound to the PRDII DNA element from the interferon-β promoter. Structure. 10, 383–391.

    Article  CAS  PubMed  Google Scholar 

  30. Romanenkov A.S., Ustyugov A.A., Zatsepin T.S., Nikulova A.A., Kolesnikov I.V., Metelev V.G., Oretskaya T.S., Kubareva E.A. 2005. Analysis of DNA–protein interactions in complexes of transcription factor NF-κB with DNA. Biochemistry (Moscow). 70 (11), 1212–1222.

    CAS  PubMed  Google Scholar 

  31. Kim Y., Ho S.O., Gassman N.R., Korlann Y., Landorf E.V., Collart F.R., Weiss S. 2008. Efficient site-specific labeling of proteins via cysteines. Bioconjug. Chem.19, 786–791.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Kapanidis A.N., Weiss S. 2002. Fluorescent probes and bioconjugation chemistries for single-molecule fluorescence analysis of biomolecules. J. Chem. Phys.117, 10953–10964.

    Article  CAS  Google Scholar 

  33. Reck-Peterson S.L., Derr N.D., Stuurman N. 2010. Imaging single molecules using total internal reflection fluorescence microscopy (TIRFM). Cold Spring Harb. Protoc.3, pdb.top73.

  34. Liu J., Hanne J., Britton B.M., Bennett J., Kim D., Lee J.B., Fishel R. 2016. Cascading MutS and MutL sliding clamps control DNA diffusion to activate mismatch repair. Nature. 539, 583–587.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Rogulin E.A., Perevyazova T.A., Zheleznaya L.A., Matvienko N.I. 2004. Plasmid pRARE as a vector for cloning to construct a superproducer of the site-specific nickase N.BspD6I. Biochemistry (Moscow). 69 (10), 1123−1127.

    CAS  PubMed  Google Scholar 

  36. Laemmli U.K. 1970. Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature.227, 680–685.

    Article  CAS  PubMed  Google Scholar 

  37. Monakhova M.V., Kubareva E.A., Romanova E.A., Semkina A.S., Naberezhnov D.S., Rao D.N., Zatsepin T.S., Oretskaya T.S. 2019. Synthesis of β‑diketone DNA derivatives for affinity modification of proteins. Russ. J. Bioorg. Chem.45 (2), 144‒154.

    Article  CAS  Google Scholar 

  38. Pingoud A., Wilson G.G., Wende W. 2014. Type II restriction endonucleases: A historical perspective and more. Nucleic Acids Res.42, 7489–527.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Bitinaite J., Wah D.A., Aggarwal A.K., Schildkraut I. 1998. FokI dimerization is required for DNA cleavage. Proc. Natl. Acad. Sci. U. S. A.95, 10570–10575.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Friedhoff P., Manelyte L., Giron-Monzon L., Winkler I., Groothuizen F.S., Sixma T.K. 2017. Use of single-cysteine variants for trapping transient states in DNA mismatch repair. Methods Enzymol.592, 77–101.

    Article  CAS  PubMed  Google Scholar 

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ACKNOWLEDGMENTS

We are grateful to T.S. Zatsepin (Skolkovo Institute of Science and Technologies, Moscow) for the oligodeoxyribonucleotides carrying the 2'-amino group and M.V. Mona-khova (Belozersky Institute of Physico-Сhemical Biology, Lomonosov Moscow State University) for the MutS preparation.

Funding

This work was supported by the Russian Science Foundation (project no. 18-74-00049).

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Authors and Affiliations

  1. Chemistry Department, Moscow State University, 119991, Moscow, Russia

    L. A. Abrosimova

  2. SABNP, University of Evry, INSERM U1204, Université Paris-Saclay, 91025, Evry, France

    A. R. Samsonova

  3. Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, 142290, Pushchino, Moscow oblast, Russia

    T. A. Perevyazova, A. K. Yunusova, R. I. Artyukh & L. A. Zheleznaya

  4. Belozersky Institute of Physico-Сhemical Biology, Moscow State University, 119991, Moscow, Russia

    E. A. Romanova, T. S. Oretskaya & E. A. Kubareva

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  1. L. A. Abrosimova
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  4. A. K. Yunusova
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Correspondence to L. A. Abrosimova.

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This work does not contain any studies involving animals or human subjects performed by any of the authors.

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Translated by T. Tkacheva

Abbreviations: dsDNA, double-stranded DNA; CD, circular dichroism; NE, nicking endonuclease; PAGE, polyacrylamide gel electrophoresis; RE, restriction endonuclease; FAM, carboxyfluorescein; PEG-Mal, polyethylene glycol maleimide; SPDP, succinimidyl 3-(2-pyridyldithio)propionate.

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Abrosimova, L.A., Samsonova, A.R., Perevyazova, T.A. et al. The Role of Cysteine Residues in the Interaction of Nicking Endonuclease BspD6I with DNA. Mol Biol 54, 599–610 (2020). https://doi.org/10.1134/S0026893320040020

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