Molecules (Apr 2022)

Reactive Acrylamide-Modified DNA Traps for Accurate Cross-Linking with Cysteine Residues in DNA–Protein Complexes Using Mismatch Repair Protein MutS as a Model

  • Mayya V. Monakhova,
  • Elena A. Kubareva,
  • Kirill K. Kolesnikov,
  • Viktor A. Anashkin,
  • Egor M. Kosaretskiy,
  • Maria I. Zvereva,
  • Elena A. Romanova,
  • Peter Friedhoff,
  • Tatiana S. Oretskaya,
  • Timofei S. Zatsepin

DOI
https://doi.org/10.3390/molecules27082438
Journal volume & issue
Vol. 27, no. 8
p. 2438

Abstract

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Covalent protein capture (cross-linking) by reactive DNA derivatives makes it possible to investigate structural features by fixing complexes at different stages of DNA–protein recognition. The most common cross-linking methods are based on reactive groups that interact with native or engineered cysteine residues. Nonetheless, high reactivity of most of such groups leads to preferential fixation of early-stage complexes or even non-selective cross-linking. We synthesised a set of DNA reagents carrying an acrylamide group attached to the C5 atom of a 2′-deoxyuridine moiety via various linkers and studied cross-linking with MutS as a model protein. MutS scans DNA for mismatches and damaged nucleobases and can form multiple non-specific complexes with DNA that may cause non-selective cross-linking. By varying the length of the linker between DNA and the acrylamide group and by changing the distance between the reactive nucleotide and a mismatch in the duplex, we showed that cross-linking occurs only if the distance between the acrylamide group and cysteine is optimal within the DNA–protein complex. Thus, acrylamide-modified DNA duplexes are excellent tools for studying DNA–protein interactions because of high selectivity of cysteine trapping.

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