1. Reactive Acrylamide-Modified DNA Traps for Accurate Cross-Linking with Cysteine Residues in DNA-Protein Complexes Using Mismatch Repair Protein MutS as a Model.
- Author
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Monakhova MV, Kubareva EA, Kolesnikov KK, Anashkin VA, Kosaretskiy EM, Zvereva MI, Romanova EA, Friedhoff P, Oretskaya TS, and Zatsepin TS
- Subjects
- Acrylamide, Base Pair Mismatch, DNA chemistry, DNA Mismatch Repair, DNA Repair, MutS DNA Mismatch-Binding Protein chemistry, MutS DNA Mismatch-Binding Protein metabolism, Proteins, Cysteine chemistry, Escherichia coli Proteins metabolism
- Abstract
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.
- Published
- 2022
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