Bypass of Mutagenic O⁶-Carboxymethylguanine DNA Adducts by Human Y- and B-Family Polymerases

Abstract

ABSTRACT:The generation of chemical alkylating agents from nitrosation of glycine and bile acid conjugates in the gastrointestinal tract is hypothesized to initiate carcinogenesis. O 6 -carboxymethylguanine (O 6 -CMG) is a product of DNA alkylation derived from nitrosated glycine. Although the tendency of the structurally related adduct O 6 -methylguanine to code for the misincoporation of TTP during DNA replication is well-established, the impact of the presence of the O 6 -CMG adduct in a DNA template on the efficiency and fidelity of translesion DNA synthesis (TLS) by human DNA polymerases (Pols) have hitherto not been described. Herein, we characterize the ability of the four human TLS Pols η, ι, κ and ζ and the replicative Pol δ to bypass O 6 -CMG in a prevalent mutational hot-spot for cancer. The results indicate that Pol η replicates past O 6 -CMG, incorporating dCMP or dAMP, whereas Pol κ exclusively performs error-free insertion and Pol ι displays the lowest fidelity. Additionally, we found that the subsequent extension step was carried out with high efficiency by TLS Pols η, κ and ζ, while Pol ι was unable to extend from a terminal mismatch. These results provide a first basis of O 6 -CMG-promoted base misincorporation by Y-and B-family polymerases potentially leading to mutational signatures associated with cancer.

Repair and translesion synthesis of O6-alkylguanine DNA lesions in human cells

“…In particular, we found significant drops in mutation frequency for these lesions in Poldeficient cells. Our observation is in accordance with in vitro studies showing a strong preference of Pol for misincorporating dTTP opposite O 6 -alkyl-dG lesions (29,72). Previous structural studies have demonstrated that, unlike Pol and Pol , which employ Watson-Crick base-pairing, Pol utilizes Hoogsteen base-pairing for DNA synthesis opposite template purines (73,74).…”

“…It is worth noting that Pol is more accurate than other TLS polymerases in bypassing branched-chain lesions. Although in vitro findings revealed that Pol is able to incorporate dCMP and dTMP efficiently opposite the straight-chain O 6 -Me-dG, a recent study showed error-free bypass of O 6 -carboxymethylguanine by human Pol Repair and replication of O 6 -alkylguanine lesions (72). These results indicate a unique nucleotide selectivity of Pol upon bypassing the highly blocking branched-chain O 6 -alkyl-dG lesions.…”

Replication studies of carboxymethylated DNA lesions in human cells

“…The induction of G→A mutation is in keeping with a recent crystal structure study of O 6 -CMdG-containing duplex DNA showing that the lesion can form base pair with thymidine through Watson–Crick base pairing (Figure 6) (32). In addition, a recent study showed that, among the several mammalian TLS polymerases tested, Pol η catalyzed the most efficient bypass and extension over O 6 -CMdG adduct in vitro (33), which differs from our finding about the lack of involvement of Pol η in bypassing the O 6 -CMdG lesion in human cells. This difference could again be attributed to the difference between replication study with a single purified polymerase in vitro and that with the entire replication machinery in cells.…”

Nanopore Sequencing Accurately Identifies the Mutagenic DNA Lesion O6‐Carboxymethyl Guanine and Reveals Its Behavior in Replication

“…While attack by these agents is thought to have low sequence specificity, recent efforts have shown that it is the selectivity of DNA repair rather than the selectivity of the initial damage that determines mutational hotspots. For example, with cisplatin and dihydropyrimidine dimers, densely packed heterochromatic regions and transcription factor binding sites are most prone to mutation . Studies of this sort highlight the need for single nucleotide resolution in sequencing DNA lesions.…”

“…[4] These can interfere with the high-fidelity transmission of genetic information [5] or lead directly to cell death [6] if they are not repaired. Alkylated DNAadducts are often carcinogenic, [7] and are formed by the attack of electrophilic alkylating agents on the nitrogen or oxygen atoms of DNAbases. [8] While attack by these agents is thought to have low sequence specificity,r ecent efforts have shown that it is the selectivity of DNArepair rather than the selectivity of the initial damage that determines mutational hotspots.F or example,w ith cisplatin [2a,7,9] and dihydropyrimidine dimers, [10] densely packed heterochromatic regions and transcription factor binding sites are most prone to mutation.…”