Peptide Sequence and Cross-Link Structure Influence Translesion Synthesis Polymerase Bypass of 5-Formylcytosine-Mediated DNA–Peptide Cross-Links
Year: 2025
Type: article
Abstract: DNA–peptide cross-links (DpCs) are generated via the proteolytic cleavage of DNA–protein cross-links (DPCs), ubiquitous DNA lesions that block DNA replication and transcription. Translesion synthesis (TLS) DNA polymerases can facilitate replication bypass of DpC adducts in either an error-free or error-prone manner. We have previously demonstrated that local DNA sequence context significantly influences hPol η -mediated replication bypass of 5-formylcytosine (5fC)-mediated DpC lesions. However, the effects of peptide sequence on the efficiency and fidelity of the TLS bypass of 5fC-mediated DpC lesions remained unknown. In the present study, model DpCs containing three different peptides (NH 2 -GGGKGLG K* GGA-COOH, NH 2 -RP K* PQQFFGLM-COOH, and NH 2 -RPKPQQF K* GLM-COOH, K* = oxy-lysine) were subjected to primer extension experiments in the presence of TLS polymerases. We found that in vitro replication of DpC-containing templates by hPol η was more efficient than that catalyzed by hPol l or hPol κ. HPLC-ESI-MS and HPLC-ESI-MS/MS analyses of hPol η primer extension products indicated that the replication bypass of DpC containing NH 2 -RP K *PQQFFGLM-COOH was more error-prone than replication of the other two DpCs, leading to targeted C → T transitions, small deletions, and untargeted mutations downstream from the lesion. Steady-state kinetics investigation of hPol η -catalyzed nucleotide incorporation opposite the DpC lesions containing three different peptides revealed that, in all cases, error-free replication was far more efficient than incorporation of incorrect nucleotides. For mutagenic bypass, the catalytic efficiency of hPol η -mediated dAMP misincorporation opposite DpC with peptide NH 2 -RP K* PQQFFGLM-COOH was higher than adenine misincorporation across from the other two DpCs and unmodified dC. These steady-state kinetic findings were further explained by molecular modeling and molecular dynamics simulations, revealing that the three different DpC lesions impose varying perturbations to the geometry of the C–G and C–A pairs at the hPol η active site. Collectively, our results reveal that the peptide sequence and conjugation chemistry of DpC lesions can influence the fidelity of lesion bypass by TLS polymerases.
Source: Chemical Research in Toxicology
Language: English
Topic: DNA Repair Mechanisms
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W4412696178