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What are DNA-protein cross-links?
What are DNA-protein cross-links?
DNA-Protein Cross-links (DPCs) are unusually bulky lesions that form when proteins are covalently trapped onto DNA. DPCs are induced by exposure to agents such as reactive oxygen species, reactive nitrogen species, aldehydes, and chemotherapy drugs. DPCs accumulate in the heart and brain tissues and are hypothesized to contribute to aging, cancer, and neurological diseases. Interestingly, DPCs involving histone proteins were recently shown to reversibly form in healthy cells as part of epigenetic regulation. Because of their considerable size and helix-distorting nature, DPCs can interfere with the progression of replication and transcription machineries and hence hamper the faithful expression of genetic information, potentially contributing to mutagenesis and carcinogenesis. However, the biological consequences and repair of DPCs remain largely elusive due to the structural complexity of the DPC lesions and the difficulties to generate chemically defined and biologically relevant DPC substrates.
Synthesis of DNA-protein cross-links
Synthesis of DNA-protein cross-links
Our laboratory has developed synthetic methodologies to form stable DPCs. We employed a reductive amination strategy where a basic amino acid, such as lysine or arginine, reacts with DNA containing aldehydes, such as 7-deaza-2-oxoethyl-dG, to form Schiff bases. These reversible cross-links are stabilized with sodium cyanoborohydride (NaCNBH3) as a reducing agent. With this methodology, we can form stable DPCs in 50-60% yields.
5‐Formylcytosine (5fC) is an endogenous DNA modification formed by oxidation of 5-methylcytosine (5mC). A unique feature of 5fC is the presence of a potentially reactive aldehyde group in its structure. Our lab has shown that 5fC bases in DNA readily form Schiff‐base conjugates with Lys side chains of nuclear proteins. Recently, we discovered a novel mechanism of epigenetic regulation involving the formation of reversible DNA-histone cross-links with 5fC. These covalent protein–DNA complexes are reversible (t1/2=1.8 h), suggesting that they contribute to transcriptional regulation and chromatin remodeling. To be further studied, these lesions must first be reduced to the hydrolytically stable amine linkage by NaCNBH3.
Two major limitations of the reductive amination approach to DPC synthesis is the cellular toxicity of NaCNBH3 and the lack of site-specificity. When employing this methodology, proteins containing multiple lysine or arginine residues result in non-specific linkages.
Development of novel methodologies for synthesis of site-specific DNA-protein cross-links
Development of novel methodologies for synthesis of site-specific DNA-protein cross-links
To address the reductive amination issues, we developed synthetic methodologies where we can easily form stable DPCs with the desired modifications at specific sites via an oxime ligation methodology. This method forms site-specific, hydrolytically stable DPCs, as well as increases the efficiency of the conjugation between DNA and proteins. Another advantage of this method is the lack of reducing agents, thus keeping the native structure of the protein undisturbed and providing a bioorthogonal strategy that can be safely employed in cells.
Using solid-phase peptide synthesis (SPPS), the unnatural amino acid, amino-oxy lysine, can be incorporated into peptides of interest. We have also incorporated this unnatural amino acid into Histone H3 via mutant F40 sortase mediated ligation. In this method, the Histone H3 tail containing amino-oxy lysine is synthesized through SPPS and enzymatically ligated to the globular domain of Histone H3 which is expressed in E. coli.
Biochemical studies using model DNA-protein Cross-Links
Biochemical studies using model DNA-protein Cross-Links
DNA-protein cross-links (DPCs) conjugated to 5fC block replication, but the corresponding DNA-peptide (DpCs) lesions induce mutagenesis during replication by translesion synthesis (TLS) polymerases. Recently, we discovered that the efficiency of replication by TLS polymerase ƞ of unmodified DNA and DpCs conjugated to 5fC is dependent on local DNA sequence context. The neighboring bases on either side of the DpC affect misincorporation of the incorrect base opposite the lesion.
Construction of site-specific nucleosome core particle DPCs to study DNA transactions
Construction of site-specific nucleosome core particle DPCs to study DNA transactions
The lysine side chains from histone proteins can undergo a series of modifications, such as acetylation, ubiquitylation, methylation, and other reversible posttranslational modifications that have an influence on chromatin structure and control of gene expression. There is currently a poor understanding on how DNA-histone cross-links affect chromatin structure, transcription and replication. We are integrating our oxime ligation methodology to form site-specific DNA-histone cross-links in a nucleosome core particle (NCP) to study these DNA transactions. This project incorporates multiple techniques in order to create the NCP, including synthesis of unnatural amino acids and nucleic acid phosphoramidites, solid-phase peptide chemistry, solid-phase oligonucleotide synthesis, sortase-mediated protein ligation, and protein expression.
PDB: 1KX5
Ubiquitin-mediated repair of DNA-protein cross-links
Ubiquitin-mediated repair of DNA-protein cross-links
Spartan (SPRTN) protease is recruited to stalled replication complexes to degrade DPCs for TLS polymerase bypass. The resulting peptide DNA adduct is repaired by nucleotide excision repair. Germline mutations in SPRTN results in Ruijs-Aalfs syndrome, a disorder in which patients exhibit accelerated aging, genome instability and early onset hepatocellular carcinoma.
Recent studies have uncovered key ubiquitin (Ub) signaling activities in both protease-based DNA repair pathways and conserved ubiquitin ligase pathway for proteasomal degradation of DNA-protein conjugate intermediates. Inhibition of the ubiquitin ligase proteasome degradation pathway is observed to lead to accumulation of DPCs and synergize with DPC-inducing chemotherapeutics to increase tumor cell apoptosis. Our lab uses mass-spectrometry, and quantitative PCR assays to identify, quantify and characterize ubiquitin-mediated DPC repair.
Methodology to identify, quantify, and characterize ubiquitin-mediated DPCs
Representative Publications:
Representative Publications:
Ji, S. et al. 5-Formylcytosine-Induced DNA–Peptide Cross-Links Reduce Transcription Efficiency, but Do Not Cause Transcription Errors in Human Cells. Journal of Biological Chemistry 2019, 294 (48), 18387–18397. Article
Ji, S. et al. Transcriptional Bypass of DNA-Protein and DNA-Peptide Conjugates by T7 RNA Polymerase. ACS Chemical Biology 2019, 14 (12), 2564–2575. Article
Ji, S., et al. 5-Formylcytosine Mediated DNA-Protein Cross-Links Block DNA Replication and Induce Mutations in Human Cells. Nucleic Acids Research 2018, 46 (13), 6455–6469. Article
Pujari, S. S., et al. Site-Specific Cross-Linking of Proteins to DNA: Via a New Bioorthogonal Approach Employing Oxime Ligation. Chemical Communications 2018, 54 (49), 6296–6299. Article
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