Exploring the Protein-DNA Interface in Living Cells with Photo-Crosslinked Peptide-Nucleotide Hybrids

• Applicant: Dr. Jakob Trendel
• Subject Area: Biochemistry
• Term: since 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 492625837

During the previous funding period I developed pioneering photo-crosslinking methodology for the study of protein-RNA interactions using high-intensity UV irradiation and mass spectrometry-based proteomics, which I applied to interrogate human and mouse RNA interactomes in response to various RNA-binding drugs. I discovered that, in addition to protein-RNA crosslinking, this new methodology could also be used for protein-DNA photo-crosslinking. I applied this novel approach to explore the proteome with physical access to DNA in human breast cancer cells, as well as changes in their DNA interactome in response to estrogen and genotoxic drugs. In-depth analysis of mass spectrometry data from these DNA interactomes revealed that they contained numerous peptides modified with a photo-crosslinked DNA nucleotide. These nucleotide-crosslinked peptides are of special interest because they provide direct evidence for a protein-DNA interaction, and pinpoint the DNA interface within the protein sequence with single amino acid resolution. In the proposed project, I will explore the protein-DNA interface with DNA-crosslinked peptides to identify protein sequence features that have physical access to the genome. In three work packages, I will i) improve the detection of nucleotide-crosslinked peptides adding an additional enrichment step, ii) apply the improved approach to explore protein-DNA interfaces across various cell lines, and iii) follow-up on potentially new DNA-binding features with AlphaFold3 and synthetic peptides. Because crosslinking only occurs at one or few sites within a protein-DNA complex, photo-crosslinks are rare and usually hard to detect. Therefore, additional titanium dioxide enrichment will be applied with the goal to eliminate non-crosslinked peptides and gain proteomic depth on the nucleotide-crosslinked ones. To catalogue as many protein-DNA interaction sites as possible and quantitatively assess their variability, this optimized workup will then be applied to human and mouse cell lines of different tissue origin, and under various perturbations. Uncharacterized protein sequence features identified in the crosslinking experiments will then be analyzed with AlphaFold3 for their ability to from stable complexes with DNA, and candidate peptide sequences synthesized for in vitro validation of DNA binding. In summary, in the proposed project I will investigate the proteome at the interface with DNA using advanced photo-crosslinking methodology, with the aim to catalogue and quantify protein sequence features that interact with the genome in living cells.

• DFG Programme: Research Grants