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DNA-protein cross-links (adducts) are formed in cellular DNA under a variety of conditions, particularly following exposure to an alpha,beta-unsaturated aldehyde, acrolein. DNA-protein cross-links are subject to repair or damage-tolerance processes. These adducts serve as substrates for proteolytic degradation, yielding DNA-peptide lesions that have been shown to be actively repaired by the nucleotide excision repair complex. Alternatively, DNA-peptide cross-links can be subjected to replication bypass. We present new evidence about the capabilities of DNA polymerases to synthesize DNA past such cross-links. DNAs were constructed with site-specific cross-links, in which either a tetrapeptide or a dodecylpeptide was covalently attached at the N (2) position of guanine via an acrolein adduct, and replication bypass assays were carried out with members of the DinB family of polymerases, human polymerase (pol) kappa, Escherichia coli pol IV, and various E. coli polymerases that do not belong to the DinB family. Pol kappa was able to catalyze both the incorporation and the extension steps with an efficiency that was qualitatively indistinguishable from control (undamaged) substrates. Fidelity was comparable on all of these substrates, suggesting that pol kappa would have a role in the low mutation frequency associated with replication of these adducts in mammalian cells. When the E. coli orthologue of pol kappa, damage-inducible DNA polymerase, pol IV, was analyzed on the same substrates, pause sites were detected opposite and three nucleotides beyond the site of the lesion, with incorporation opposite the lesion being accurate. In contrast, neither E. coli replicative polymerase, pol III, nor E. coli damage-inducible polymerases, pol II and pol V, could efficiently incorporate a nucleotide opposite the DNA-peptide cross-links. Consistent with a role for pol IV in tolerance of these lesions, the replication efficiency of DNAs containing DNA-peptide cross-links was greatly reduced in pol IV-deficient cells. Collectively, these data indicate an important role for the DinB family of polymerases in tolerance mechanisms of N (2)-guanine-linked DNA-peptide cross-links.