Error-prone translesion synthesis past DNA-peptide crosslinks conjugated to the major groove of DNA via C5 of thymidine [Enzymology]

November 12th, 2014 by Wickramaratne, S., Boldry, E. J., Buehler, C., Wang, Y.-C., DiStefano, M. D., Tretyakova, N. Y.

DNA-protein crosslinks (DPCs) are exceptionally bulky, structurally diverse DNA adducts formed in cells upon exposure to endogenous and exogenous bis-electrophiles, reactive oxygen species, and ionizing radiation. If not repaired, DPCs can induce toxicity and mutations. It has been proposed that the protein component of a DPC is proteolytically degraded, giving rise to smaller DNA-peptide conjugates, which can be subject to nucleotide excision repair and replication bypass. In the present study, polymerase bypass of model DNA-peptide conjugates structurally analogous to the lesions induced by reactive oxygen species and DNA methyltransferase inhibitors was examined. DNA oligomers containing site-specific DNA-peptide conjugates were generated by copper-catalyzed [3+2] Huisgen cycloaddition between an alkyne functionalized C5-thymidine in DNA and an azide-containing 10-mer peptide. The resulting DNA-peptide conjugates were subjected to steady-state kinetic experiments in the presence of recombinant human lesion bypass polymerases κ and η, followed by PAGE-based assays to determine the catalytic efficiency and the misinsertion frequency opposite the lesion. We found that hPol κ and η can incorporate A, G, C, or T opposite the C5-dT-conjugated DNA-peptide conjugates, while hPol η preferentially inserts G opposite the lesion. Furthermore, HPLC-ESI--MS/MS sequencing of the extension products has revealed that postlesion synthesis was highly error prone, resulting in mutations opposite the adducted site or at the +1 position from the adduct and multiple deletions. Collectively, our results indicate that replication bypass of peptides conjugated to the C-5 position of thymine by human TLS polymerases leads to large numbers of base substitution and frameshift mutations.