ArticleAnalytical chemistry2021
High-Resolution Mapping of Amino Acid Residues in DNA-Protein Cross-Links Enabled by Ribonucleotide-Containing DNA.
Article in Analytical chemistry, 2021. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 7 papers.
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Who cites it
7 citing papers in PubMed, 12 citations in OpenAlex.
- Mitochondrial transcription factor A (TFAM) has 5'-deoxyribose phosphate lyase activity in vitro.DNA repair · 2024Article
- 35 Years of TFAM Research: Old Protein, New Puzzles.Biology · 2023Review
- Key Amino Acid Residues of Mitochondrial Transcription Factor A Synergize with Abasic (AP) Site Dynamics To Facilitate AP-Lyase Reactions.ACS chemical biology · 2023Article
- DNA-protein cross-links between abasic DNA damage and mitochondrial transcription factor A (TFAM).Nucleic acids research · 2023Article
- Mass Spectrometry for Assessing Protein-Nucleic Acid Interactions.Analytical chemistry · 2023Review
- Article
- Facile preparation of model DNA interstrand cross-link repair intermediates using ribonucleotide-containing DNA.DNA repair · 2022Article
Corrections and comments
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Authors and funding
4 authors at 1 institution in 1 country.
Funding
Abstract
DNA-protein cross-links have broad applications in mapping DNA-protein interactions and provide structural insights into macromolecular structures. However, high-resolution mapping of DNA-interacting amino acid residues with tandem mass spectrometry remains challenging due to difficulties in sample preparation and data analysis. Herein, we developed a method for identifying cross-linking amino residues in DNA-protein cross-links at single amino acid resolution. We leveraged the alkaline lability of ribonucleotides and designed ribonucleotide-containing DNA to produce structurally defined nucleic acid-peptide cross-links under our optimized ribonucleotide cleavage conditions. The structurally defined oligonucleotide-peptide heteroconjugates improved ionization, reduced the database search space, and facilitated the identification of cross-linking residues in peptides. We applied the workflow to identifying abasic (AP) site-interacting residues in human mitochondrial transcription factor A (TFAM)-DNA cross-links. With sub-nmol sample input, we obtained high-quality fragmentation spectra for nucleic acid-peptide cross-links and identified 14 cross-linked lysine residues with the home-built AP_CrosslinkFinder program. Semi-quantification based on integrated peak areas revealed that K186 of TFAM is the major cross-linking residue, consistent with K186 being the closest (to the AP modification) lysine residue in solved TFAM:DNA crystal structures. Additional cross-linking lysine residues (K69, K76, K136, K154) support the dynamic characteristics of TFAM:DNA complexes. Overall, our combined workflow using ribonucleotide as a chemically cleavable DNA modification together with optimized sample preparation and data analysis offers a simple yet powerful approach for mapping cross-linking sites in DNA-protein cross-links. The method is amendable to other chemical or photo-cross-linking systems and can be extended to complex biological samples.
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Registered trials
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