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EcoRII Restriction Endonuclease Forms Specific Contacts to the Bases of Its Target Sequence Flipped from DNA in a Transition Complex with Photoactivatable Substrates

Russian Journal of Bioorganic Chemistry volume 47, pages 367–375 (2021)Cite this article

Abstract

The photoactivatable modified oligonucleotides were used to investigate direct contacts formed by the type IIE EcoRII restriction endonuclease and the T/A bases of its recognition site (5'-CCT/AGG). EcoRII dimer consists of a central catalytic core, made of two C-terminal endonuclease-like domains (EcoRII-C) from different subunits, and two N-terminal effector DNA binding domains (EcoRII-N). According to co-crystal structure of isolated EcoRII-C with DNA catalytic dimer EcoRII-C flips nucleotides of the central T/A pair into the enzyme binding pockets. Нere, photocross-linking technique was used to investigate the direct contacts formed by extrahelical T/A bases in the protein pockets of full-length EcoRII within the pre-reactive EcoRII–DNA complex obtained in the presence of Ca2+ in solution. Photoreactive zero-length agent 5-iodo-2'-deoxyuridine (IdU) was introduced as single substituent into the central T/A position of EcoRII recognition site or into the flanking nucleotide sequences of 14-mer DNA substrate. The substitution of only dT or dA residues in EcoRII recognition site resulted in formation of photocross-links upon irradiation only in the presence of Ca2+. Proteolytic digestion of the enzyme-oligonucleotide conjugates followed by MALDI-MS analysis have allowed to identify the 224VEYD227 EcoRII region involved in the formation of the cross-links. This region belongs to the central part of H-10 α-helix. Y226 residue was suggested to form cross-link with T or A bases of EcoRII site replaced by IdU within the pre-reactive complex. The flipped base pair protein pockets of EcoRII seem to accommodate equally well both A and T bases of the DNA substrate. Altogether, IdU-containing photoactivatable DNA substrates have allowed to trap the flipped bases in complex with full-length EcoRII before DNA cleavage in the solution and to identify direct enzyme–DNA contacts important for high specificity of EcoRII for the Т/A nucleotides providing a highly specific cleavage reaction.

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ACKNOWLEDGMENTS

We are grateful to T. Zatsepin for recording of MALDI mass spectra.

Funding

This work was supported by the Russian Foundation for Fundamental Investigations under Grant no. 19-04-00533.

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Authors and Affiliations

  1. Moscow State University, Department of Chemistry, 119234, Moscow, Russia

    O. V. Kirsanova, F. V. Subach, A. G. Loiko & E. S. Gromova

  2. National Research Center Kurchatov Institute, 123182, Moscow, Russia

    F. V. Subach

  3. CIBER-BBN, Institute for Advanced Chemistry of Catalonia (IQAC), Spanish Council for Scientific Research (CSIC), Jordi Girona 18-26, 08034, Barcelona, Spain

    R. I. Eritja

Authors
  1. O. V. Kirsanova
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  2. F. V. Subach
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  3. A. G. Loiko
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  4. R. I. Eritja
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  5. E. S. Gromova
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Correspondence to E. S. Gromova.

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

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Abbreviations: DTT, dithiothreitol; IBA, o-iodosobenzoic acid; IdC, 5-iodo-2'-deoxycytidine; IdU, 5-iodo-2'-deoxyuridine; NCS, N-chlorosuccinimide; NTCBA, 2-nitro-5-thiocyanobenzoic acid; ON, oligodeoxynucleotide; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecylsulfate; Tris, tris(hydroxymethyl)aminomethane.

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Kirsanova, O.V., Subach, F.V., Loiko, A.G. et al. EcoRII Restriction Endonuclease Forms Specific Contacts to the Bases of Its Target Sequence Flipped from DNA in a Transition Complex with Photoactivatable Substrates. Russ J Bioorg Chem 47, 367–375 (2021). https://doi.org/10.1134/S106816202102014X

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  • DOI: https://doi.org/10.1134/S106816202102014X

Keywords:

  • restriction endonuclease
  • DNA–protein interactions
  • photocross-linking
  • 5-iodo-2'-deoxyuridine
  • base flipping mechanism