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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 (gamma-hydroxypropano-dA or gamma-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 gamma-hydroxypropano-dA-mediated crosslink induced mutations at only approximately 0.4%. In contrast, replication bypass of the gamma-hydroxypropano-dG-mediated crosslink resulted in mutations at the site of modification at an overall frequency of approximately 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 gamma-hydroxypropano-dG caused an increase in mutation frequencies relative to gamma-hydroxypropano-dG, similar modification of gamma-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.