Reactive acrylamide-modified DNA traps for accurate cross-linking with cysteine residues in DNA–protein complexes using mismatch repair protein MutS as a model

Monakhova, M.V.; Kubareva, Е.A.; Kolesnikov, K.K.; Anashkin, V.A.; Kosaretskiy, Е.M.; Zvereva, M.I.; Romanova, E.A.; Friedhoff, P.; Oretskaya, T.S.; Zatsepin, T.S.

Авторы: Monakhova M.V., Kubareva Е.A., Kolesnikov K.K., Anashkin V.A., Kosaretskiy Е.M., Zvereva M.I., Romanova E.A., Friedhoff P., Oretskaya T.S., Zatsepin T.S.
Журнал: Molecules
Том: 27
Номер: 8
Год издания: 2022
Издательство: MDPI
Местоположение издательства: Basel, Switzerland
Первая страница: 2438
DOI: 10.3390/molecules27082438
Аннотация: Covalent protein capture (cross-linking) by reactive DNA derivatives makes it possible toinvestigate 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 orengineered cysteine residues. Nonetheless, high reactivity of most of such groups leads to preferentialfixation of early-stage complexes or even non-selective cross-linking. We synthesised a set of DNAreagents carrying an acrylamide group attached to the C5 atom of a 2′-deoxyuridine moiety viavarious linkers and studied cross-linking with MutS as a model protein. MutS scans DNA formismatches and damaged nucleobases and can form multiple non-specific complexes with DNAthat may cause non-selective cross-linking. By varying the length of the linker between DNA andthe acrylamide group and by changing the distance between the reactive nucleotide and a mismatchin the duplex, we showed that cross-linking occurs only if the distance between the acrylamidegroup and cysteine is optimal within the DNA–protein complex. Thus, acrylamide-modified DNAduplexes are excellent tools for studying DNA–protein interactions because of high selectivity ofcysteine trapping.
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Reactive acrylamide-modified DNA traps for accurate cross-linking with cysteine residues in DNA–protein complexes using mismatch repair protein MutS as a modelстатья

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