Peptide Sequence and Cross-Link Structure Influence Translesion Synthesis Polymerase Bypass of 5-Formylcytosine-Mediated DNA-Peptide Cross-Links.

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 (NH₂-GGGKGLGK*GGA-COOH, NH₂-RPK*PQQFFGLM-COOH, and NH₂-RPKPQQFK*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 NH₂-RPK*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 NH₂-RPK*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.