Error-prone translesion synthesis past DNA-peptide cross-links conjugated to the major groove of DNA via C5 of thymidine.
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.
From the Masonic Cancer Center, Departments of Medicinal Chemistry and Chemistry, University of Minnesota, Minneapolis, Minnesota 55455.
Susith
S
Wickramaratne
From the Masonic Cancer Center, Departments of Medicinal Chemistry and.
Emily J
EJ
Boldry
Chemistry, University of Minnesota, Minneapolis, Minnesota 55455.
Charles
C
Buehler
Chemistry, University of Minnesota, Minneapolis, Minnesota 55455.
Yen-Chih
YC
Wang
From the Masonic Cancer Center, Departments of Medicinal Chemistry and Chemistry, University of Minnesota, Minneapolis, Minnesota 55455.
Mark D
MD
Distefano
From the Masonic Cancer Center, Departments of Medicinal Chemistry and trety001@umn.edu.
Natalia Y
NY
Tretyakova
D057930:Click Chemistry; D018736:DNA Adducts / Q000737:chemistry / Q000378:metabolism*; D004249:DNA Damage / Q000235:genetics; D004260:DNA Repair / Q000235:genetics; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D009369:Neoplasms / Q000378:metabolism* / Q000473:pathology; D010455:Peptides / Q000737:chemistry / Q000235:genetics*; D017382:Reactive Oxygen Species / Q000378:metabolism; D053719:Tandem Mass Spectrometry; D013936:Thymidine / Q000737:chemistry / Q000235:genetics*
D018736:DNA Adducts; D010455:Peptides; D017382:Reactive Oxygen Species; D004259:DNA-Directed DNA Polymerase; D013936:Thymidine
Cancer; Click Chemistry; DNA Damage; DNA Polymerase; DNA-Protein Conjugates; Mass Spectrometry (MS); Mutations; Steady-state Kinetics; Translesion Synthesis