Reactive Acrylamide-Modified DNA Traps for Accurate Cross-Linking with Cysteine Residues in DNA–Protein Complexes Using Mismatch Repair Protein MutS as a Model
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 cys...
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MDPI AG
2022-04-01
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| author | 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 |
| author_facet | 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 |
| author_sort | Mayya V. Monakhova |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-09T04:22:12Z |
| format | Article |
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| institution | Directory Open Access Journal |
| issn | 1420-3049 |
| language | English |
| last_indexed | 2024-03-09T04:22:12Z |
| publishDate | 2022-04-01 |
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| record_format | Article |
| series | Molecules |
| spelling | doaj.art-a814b76993eb444783e3801142ca0d1b2023-12-03T13:46:10ZengMDPI AGMolecules1420-30492022-04-01278243810.3390/molecules27082438Reactive Acrylamide-Modified DNA Traps for Accurate Cross-Linking with Cysteine Residues in DNA–Protein Complexes Using Mismatch Repair Protein MutS as a ModelMayya V. Monakhova0Elena A. Kubareva1Kirill K. Kolesnikov2Viktor A. Anashkin3Egor M. Kosaretskiy4Maria I. Zvereva5Elena A. Romanova6Peter Friedhoff7Tatiana S. Oretskaya8Timofei S. Zatsepin9A.N. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Leninskye Gory 1, 119991 Moscow, RussiaA.N. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Leninskye Gory 1, 119991 Moscow, RussiaDepartment of Chemistry, Lomonosov Moscow State University, Leninskye Gory 1, 119991 Moscow, RussiaA.N. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Leninskye Gory 1, 119991 Moscow, RussiaDepartment of Bioengineering and Bioinformatics, Lomonosov Moscow State University, Leninskye Gory 1, 119991 Moscow, RussiaDepartment of Chemistry, Lomonosov Moscow State University, Leninskye Gory 1, 119991 Moscow, RussiaA.N. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Leninskye Gory 1, 119991 Moscow, RussiaInstitute for Biochemistry, FB 08, Justus Liebig University, Heinrich-Buff-Ring 17, D-35392 Giessen, GermanyA.N. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Leninskye Gory 1, 119991 Moscow, RussiaDepartment of Chemistry, Lomonosov Moscow State University, Leninskye Gory 1, 119991 Moscow, RussiaCovalent 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.https://www.mdpi.com/1420-3049/27/8/2438regioselectivityDNA modificationDNA–protein complexmodified oligonucleotidecrosslinkingDNA mismatch repair |
| spellingShingle | 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 Reactive Acrylamide-Modified DNA Traps for Accurate Cross-Linking with Cysteine Residues in DNA–Protein Complexes Using Mismatch Repair Protein MutS as a Model Molecules regioselectivity DNA modification DNA–protein complex modified oligonucleotide crosslinking DNA mismatch repair |
| title | Reactive Acrylamide-Modified DNA Traps for Accurate Cross-Linking with Cysteine Residues in DNA–Protein Complexes Using Mismatch Repair Protein MutS as a Model |
| title_full | Reactive Acrylamide-Modified DNA Traps for Accurate Cross-Linking with Cysteine Residues in DNA–Protein Complexes Using Mismatch Repair Protein MutS as a Model |
| title_fullStr | Reactive Acrylamide-Modified DNA Traps for Accurate Cross-Linking with Cysteine Residues in DNA–Protein Complexes Using Mismatch Repair Protein MutS as a Model |
| title_full_unstemmed | Reactive Acrylamide-Modified DNA Traps for Accurate Cross-Linking with Cysteine Residues in DNA–Protein Complexes Using Mismatch Repair Protein MutS as a Model |
| title_short | Reactive Acrylamide-Modified DNA Traps for Accurate Cross-Linking with Cysteine Residues in DNA–Protein Complexes Using Mismatch Repair Protein MutS as a Model |
| title_sort | reactive acrylamide modified dna traps for accurate cross linking with cysteine residues in dna protein complexes using mismatch repair protein muts as a model |
| topic | regioselectivity DNA modification DNA–protein complex modified oligonucleotide crosslinking DNA mismatch repair |
| url | https://www.mdpi.com/1420-3049/27/8/2438 |
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