Generation of site-specific DNA-polypeptide cross-links mediated by Schiff base chemistry: A novel strategy to investigate the cellular pathways that repair DNA-protein cross-link damage

DNA-protein cross-links (DPCs) are induced in biological systems upon exposure to several chemical and physical agents, many of which are known or suspected carcinogens. The resulting covalent linkages are unique to each agent, leading to cross-links at specific chemical functions on both DNA and protein. A survey of the current literature indicates that excision repair participates in the active removal of DPCs formed by only a subset of the known DPC-inducing agents; thus, a unified repair scheme has not emerged for this type of lesion. This observation suggests that a detailed understanding of the chemical and structural attributes of a particular DPC is critical in elucidating the pathways that serve to repair the lesion.; The DNA repair proteins termed “bifunctional glycosylase/AP lyase” enzymes perform DNA strand incision as part of their catalytic activity, and this reaction is initiated at the ring-open aldehydic function at an abasic site. A protein amine serves as the reactive nucleophile to form a transient protein-DNA Schiff base intermediate, which can be isolated as a stable covalent species under reducing conditions. Several polypeptides of defined composition are able to catalyze this same reaction and can be cross-linked to an abasic site in a similar manner. In addition, a number of aldehyde-derived DNA adducts form ring-open aldehydic tautomers in duplex DNA that react in a fashion analogous to the aldehydic function at an abasic site. Thus, utilizing AP site-containing DNA and site-specifically adducted DNAs as starting materials, a chemical trapping methodology was used to generate an array of model substrates containing site-specific DNA-peptide and DNA-protein cross-links.; Recombinant proteins from Bacillus caldotenax and Thermotoga maritima were obtained to determine the capacity of an in vitro nucleotide excision repair system to initiate repair on the DNA-polypeptide substrates. The coordinated action of the repair proteins excised a repair “patch” of DNA (containing the cross-linked damaged DNA strand) at comparable efficiency for different sites of covalent linkage on the DNA. Small DNA-peptide cross-links were excised more efficiently than larger DNA-protein cross-links, suggesting that the repair of certain DPCs includes the targeted proteolysis of proteins that have become covalently cross-linked to DNA.