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 O 6 -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.

DNA polymerases η and κ bypass N2-guanine-O6-alkylguanine DNA alkyltransferase cross-linked DNA-peptides

“…As discussed earlier, in steady-state kinetics the extent of misincorporation observed opposite the N 2 -dG-15-mer peptide cross-link was less than for dG (Table 1). In contrast, our own studies with N 6 -dA-oligonucleotide-peptide cross-links showed 37% of overall misincorporation by hpol η (39).…”

“…The (38,39) and 36-mer peptides were J o u r n a l P r e -p r o o f treated with O-(mesitylsulfonyl)hydroxylamine (MSH) to convert the cysteine residue to dehydroalanine (dha) (Fig. S1) (40).…”

“…Human TLS pol and pol mediated bypass of N 2 -dG-peptide cross-links Full-length extension and single nucleotide incorporation assays were carried out with two major human (h) TLS pols, η and κ. Preliminary studies with shorter peptides (3-, 5-, 7-, and 11-mers) bound at the N6 atom of dA, prepared using the same dehydroalanine strategy (39,40), were bypassed readily by these two polymerases and we focused our work on the AGT active site 15-mer peptide bound at the N2 atom of dG, in that it was more informative of longer peptides that might be present in cells. In the course of our work, we extended the studies to a 36-mer AGT active site peptide cross-link at the N2 atom of dG, although we did not repeat all of the miscoding studies.…”

“…As observed earlier in our laboratory and by others (e.g., ref. (39)), hpol η is prone to misincorporation at high dNTP concentrations and to insert multiple copies of a single dNTP. hpol η preferentially added dCTP across from (unmodified) dG, with the incorporation preference dCTP > dATP > dTTP > dGTP (Fig.…”

Randomized Controlled Trial of DDS for Covid-19 ARDS

“…DDS has the following potential: DDS can be used along with oral therapeutics for physically obstructing the replication of DNA in in ammasomes. Proteins covalently attached to DNA are common and impose physical obstacles to DNA replication, repair, transcription, and recombination[61] and large DNA-protein crosslinks can be cleaved into DNA-peptide crosslinks; these smaller fragments also disrupt normal replication [62]. (Fig.…”