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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

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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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