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

Abstract

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 (k_cat/K_m) 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.

Keywords: DNA alkylation; DNA cross-ink; DNA cross-link repair; DNA damage; DNA polymerase; DNA replication; DNA–protein interaction.

Figure 1

AGT-DNA cross-links induced by ethylene dibromide (50).

Figure 2

Synthetic strategy for 15-mer peptide cross-linked at the N6 atom of dA in an oligonucleotide.

Figure 3

MALDI mass spectrum of oxidized 15-mer peptide-N⁶-dA oligonucleotide cross-link. Expected mass [M + H]⁺ 7443.8107, observed mass [M + H]⁺ 7443.6817.

Figure 4

Full-length extension by hPol η, ɩ, and κ in the presence of all four dNTPs.A, 12-mer primer and 19-mer template DNA sequences, where X is dA or 15-mer peptide cross-linked at N⁶-dA. Reactions were done in the presence of: (B) 20 nM hPol η; (C) 40 nM hPol ɩ; and (D) 20 nM hPol κ. All experiments were done at 37 °C for 0, 5, 10, 20, 60, and 120 min (i.e., lanes, 1–6, 7–12, 13–18, 19–24, 25–30, 31–36). P, FAM-labeled 12-mer DNA primer.

Figure 5

Single nucleotide insertion by hPol η, ɩ, and κ.A, 14-mer primer and 19-mer template sequences, where X is dA or N⁶-dA-peptide. Reactions were done in the presence of: (B) 5 nM hPol η; (C) 5 nM hPol ɩ; and (D) 5 nM hPol κ. All experiments were done at 37 °C for 0, 5, 10, and 30 min. Lanes 2–4 and 14–16, dATP; lanes 5–7 and 17–19, dTTP; lanes 8–10 and 20–22, dCTP; lanes 11–13 and 23–25, dGTP. P, FAM-labeled 14-mer DNA primer.

Figure 6

Steady-state kinetic analysis of dTTP insertion by hPol η, ɩ, and κ.A, 14-mer primer and 19-mer DNA template sequences, where X is dA or N⁶-dA-peptide. Reactions were done at 37 °C using hPol η: (B) 0.09 nM and (C) 0.1 nM; hPol ɩ: (D) 0.01 and (E) 2 nM; hPol κ: (F) 2 nM and (G) 2.5 nM. Varying concentrations of dTTP were used: (B) 0–15 μM; (C) 0–90 μM; (D) 0–240 μM; (E) 0–500 μM; (F) 0–10 μM; (G) 0–2000 μM. Reactions were carried out for: (F) 8 min; (D and E) 10 min; (B and C) 12 min; (G) 21 min. P, FAM-labeled 14-mer DNA primer. Data points shown are means ± SD.

Figure 7

Extracted ion chromatogram and CID spectrum of m/z 944.27 (−3, RT 4.44) for 15-mer peptide crosslinked to DNA. A, extracted ion chromatogram; B, CID spectrum of m/z 944.27 ion.