DNA-Peptide Crosslinks Placed at Codon 249 of the p53 Tumor Suppressor Gene Induce G to A Transitions in Human Cells.

Anticancer nitrogen mustard drugs including cyclophosphamide, mechlorethamine and chlorambucil induce covalent DNA-protein cross-links (DPCs) by sequentially alkylating the N7 position of guanine in DNA and amino acid residues within proteins. In cells, DPCs are proteolytically processed to yield corresponding peptide-DNA cross-links (DpCs), allowing polymerase bypass. In this study, site-specific, hydrolytically stable DNA lesions resembling nitrogen mustard-induced DpCs were generated via oxime ligation of an 11-mer peptide derived from histone H4 (NH2-GGGK*GLGKGGA, K*=oxy-lysine) to 7-deaza-7-(2,3-dihydroprop-1-yl)-dG (DHP) in DNA. DNA sequence was derived from codon 249 of the p53 tumor suppressor gene, a common mutational hotspot in tumors. Translesion synthesis (TLS) experiments of DpC containing plasmids in human HEK 293T cells revealed that 7-deaza-DpC lesion was bypassed most efficiently in wild-type cells (83%) with lower efficiency in Pol ι KO (53%), Pol η KO (59%), and Pol κ KO (63%) cells, with Pol ζ KO unaffected (76%). Mutation frequency for polymerase bypass of 7-deaza-DpC (~9%) was significantly higher than for 7-deaza-DHP-dG control (~5%). Pol η and Pol ι knockouts led to increased error rates, suggesting they play a role in error-free bypass of DpCs. In contrast Pol ζ deficiency reduced mutation rates, implying that it plays a role in error-prone bypass.