High-Resolution Mapping of Amino Acid Residues in DNA–Protein Cross-Links Enabled by Ribonucleotide-Containing DNA
journal contributionposted on 24.09.2021, 17:03 by Jin Tang, Wenxin Zhao, Nathan G. Hendricks, Linlin Zhao
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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quality fragmentation spectraprovide structural insightsdatabase search spacecomplex biological samplesbuilt ap_crosslinkfinder programnmol sample inputamino acid residueslinked lysine residuesoptimized sample preparationlinking amino residuesidentified 14 crossdata analysis offerslinking lysine residuesdna crystal structuressample preparationlinking residuesdata analysislysine residueinteracting residuesmacromolecular structureslinking systemslinking siteslinking residueresolution mappingquantification basedmapping crossmajor crossidentifying crossidentifying abasicdynamic characteristicsdna complexescontaining dnabroad applicationsalkaline labilityadditional cross