Enzymatic bypass of an N6-deoxyadenosine DNA–ethylene dibromide–peptide cross-link by translesion DNA polymerases

• Published in: The Journal of biological chemistry 2021-01, Vol.296, p.100444, Article 100444
• Main Authors: Ghodke, Pratibha P., Gonzalez-Vasquez, Gabriela, Wang, Hui, Johnson, Kevin M., Sedgeman, Carl A., Guengerich, F. Peter
• Format: Article
• Language: English
• Subjects: DNA alkylation; DNA cross-ink; DNA cross-link repair; DNA damage; DNA polymerase; DNA replication; DNA–protein interaction
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/j.jbc.2021.100444

Unrepaired DNA–protein cross-links, due to their bulky nature, can stall replication forks and result in genome instability. Large DNA–protein cross-links can be cleaved into DNA–peptide cross-links, but the extent to which these smaller fragments disrupt normal replication is not clear. Ethylene dibromide (1,2-dibromoethane) is a known carcinogen that can cross-link the repair protein O6-alkylguanine-DNA alkyltransferase (AGT) to the N6 position of deoxyadenosine (dA) in DNA, as well as four other positions in DNA. We investigated the effect of a 15-mer peptide from the active site of AGT, cross-linked to the N6 position of dA, on DNA replication by human translesion synthesis DNA polymerases (Pols) η, ⍳, and κ. The peptide–DNA cross-link was bypassed by the three polymerases at different rates. In steady-state kinetics, the specificity constant (kcat/Km) for incorporation of the correct nucleotide opposite to the adduct decreased by 220-fold with Pol κ, tenfold with pol η, and not at all with Pol ⍳. Pol η incorporated all four nucleotides across from the lesion, with the preference dT > dC > dA > dG, while Pol ⍳ and κ only incorporated the correct nucleotide. However, LC-MS/MS analysis of the primer-template extension product revealed error-free bypass of the cross-linked 15-mer peptide by Pol η. We conclude that a bulky 15-mer peptide cross-linked to the N6 position of dA can retard polymerization and cause miscoding but that overall fidelity is not compromised because only correct pairs are extended.