Mutagenic potential of DNA–peptide crosslinks mediated by acrolein-derived DNA adducts

Abstract

Current data suggest that DNA–peptide crosslinks are formed in cellular DNA as likely intermediates in the repair of DNA–protein crosslinks. In addition, a number of naturally occurring peptides are known to efficiently conjugate with DNA, particularly through the formation of Schiff-base complexes at aldehydic DNA adducts and abasic DNA sites. Since the potential role of DNA–peptide crosslinks in promoting mutagenesis is not well elucidated, here we report on the mutagenic properties of Schiff-base-mediated DNA–peptide crosslinks in mammalian cells. Site-specific DNA–peptide crosslinks were generated by covalently trapping a lysine-tryptophan-lysine-lysine peptide to the N 6 position of deoxyadenosine (dA) or the N 2 position of deoxyguanosine (dG) via the aldehydic forms of acrolein-derived DNA adducts (γ-hydroxypropano-dA or γ-hydroxypropano-dG, respectively). In order to evaluate the potential of DNA–peptide crosslinks to promote mutagenesis, we inserted the modified oligodeoxynucleotides into a single-stranded pMS2 shuttle vector, replicated these vectors in simian kidney (COS-7) cells and tested the progeny DNAs for mutations. Mutagenic analyses revealed that at the site of modification, the γ-hydroxypropano-dA-mediated crosslink induced mutations at only ∼0.4%. In contrast, replication bypass of the γ-hydroxypropano-dG-mediated crosslink resulted in mutations at the site of modification at an overall frequency of ∼8.4%. Among the types of mutations observed, single base substitutions were most common, with a prevalence of G to T transversions. Interestingly, while covalent attachment of lysine-tryptophan-lysine-lysine at γ-hydroxypropano-dG caused an increase in mutation frequencies relative to γ-hydroxypropano-dG, similar modification of γ-hydroxypropano-dA resulted in decreased levels of mutations. Thus, certain DNA–peptide crosslinks can be mutagenic, and their potential to cause mutations depends on the site of peptide attachment. We propose that in order to avoid error-prone replication, proteolytic degradation of proteins covalently attached to DNA and subsequent steps of DNA repair should be tightly coordinated.