Abstract
DNA-protein cross-links (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 this 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 cyclo-addition 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 human polymerase κ and η can incorporate A, G, C, or T opposite the C5-dT-conjugated DNA-peptide conjugates, whereas human polymerase η preferentially inserts G opposite the lesion. Furthermore, HPLC-ESI(-)-MS/MS sequencing of the extension products has revealed that post-lesion 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 C5 position of thymine by human translesion synthesis polymerases leads to large numbers of base substitution and frameshift mutations.
Highlights
DNA-protein conjugates can be induced by reactive oxygen species and proteolytically cleaved to the corresponding peptide conjugates
Other major MS peaks at m/z 942.15, 934.15, and 922.14 corresponded to the doubly charged ions of 5Ј-pT C_G TGA-3Ј, 5Ј-pT C_A TGA-3Ј, and 5Ј-pT C_C TGA-3Ј (Table 2 and Figs. 6 and 7). These results indicate that replication bypass of dT-peptide conjugates by TLS polymerases is highly error-prone and that the extension products vary dramatically depending on DNA polymerase involved
DNA-protein cross-links (DPCs) are a ubiquitous form of DNA damage found in normal cells as a result of exposure to endogenous and exogenous reactive species [1, 5]
Summary
DNA-protein conjugates can be induced by reactive oxygen species and proteolytically cleaved to the corresponding peptide conjugates. DNA-protein cross-links (DPCs) are exceptionally bulky DNA lesions that form when cellular proteins become covalently trapped on DNA strands in the presence of exogenous and endogenous bis-electrophiles, ionizing radiation, and free radicals [1] Because of their unusual size, significant heterogeneity, and the ability to disrupt normal DNA-protein interactions [2,3,4,5,6], DPCs are hypothesized to block DNA replication, transcription, and repair and to induce cytotoxic and mutagenic effects [7,8,9]. Our results provide evidence for the highly error-prone nature of replication past C5-dT-peptide adducts by hpol and , which gives rise to the large numbers of base substitutions and frameshift mutations
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