High-Resolution Mapping of Amino Acid Residues in DNA–Protein Cross-Links Enabled by Ribonucleotide-Containing DNA

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 crosslinks at single amino acid resolution. We leveraged the alkaline lability of ribonucleotides and designed ribonucleotide-containing DNA to produce structurally defined nucleic acid−peptide crosslinks 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 highquality 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 crosslinking 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.

DNA–protein cross-links between abasic DNA damage and mitochondrial transcription factor A (TFAM)

“…The mixture was incubated at 37°C overnight to convert DNA-peptide cross-links to nucleoside-peptide cross-links. The sample was analyzed by LC–MS/MS, as described previously ( 24 ). The data analysis was performed with AP_CrosslinkFinder, followed by manual verification of cross-linked residues and annotation of the MS/MS spectra.…”

DNA–Protein Cross-Links Formed by Reacting Lysine with Apurinic/Apyrimidinic Sites in DNA and Human Cells: Quantitative Analysis by Liquid Chromatography–Tandem Mass Spectrometry Coupled with Stable Isotope Dilution

“…22,40 Mass spectrometry-based methods have significantly improved analytical sensitivity and selectivity in DPC studies. 54 For example, Swenberg and co-workers developed an LC−MS/MS method for the quantitative analysis of formaldehyde-induced DPCs in formaldehyde-exposed cells. 21 We recently also developed an LC−MS/MS coupled with a stable isotope dilution method for rigorous quantitation of DPCs formed by reacting AP sites with cysteine residues in proteins.…”

“…The chemical stability of the AP site-Lys-cross-linked DPC product has also been a concern in DPC studies, where the imine adduct is chemically reduced to amine prior to gel electrophoretic analysis. , However, as was evident by our HPLC purification of the standards and the method validation experiment using synthesized and purified Fmoc-Lys-dR, dR-Lys cross-linked peptide ( 1 ), and oligodeoxyribonucleotide ( 2 ), the DPC product was stable during both the sample workup (Figure S6) and instrumental analysis processes. Subsequent LC–MS/MS analysis targeted the imine adduct without chemical reduction.…”

“…Studies of DPCs have been predominantly descriptive in nature, as have earlier studies of AP site-Lys cross-links using gel electrophoretic detection. , Mass spectrometry-based methods have significantly improved analytical sensitivity and selectivity in DPC studies . For example, Swenberg and co-workers developed an LC–MS/MS method for the quantitative analysis of formaldehyde-induced DPCs in formaldehyde-exposed cells .…”

Mass Spectrometry for Assessing Protein–Nucleic Acid Interactions

“…Using LC-MS/MS, they uncovered 17 unique K MP sites at 57 lysine residues throughout the four core histones isolated from HeLa cells treated with bleomycin. Moreover, Tang et al 56 developed a MS-based method to identify cross-linking sites in abasic site-elicited DPC at single-amino acid resolution. They applied this method to examine TFAM−abasic site cross-links and revealed several lysine residues involved in TFAM−abasic site interactions.…”