Replication studies of carboxymethylated DNA lesions in human cells

Abstract

Metabolic activation of some N-nitroso compounds (NOCs), an important class of DNA damaging agents, can induce the carboxymethylation of nucleobases in DNA. Very little was previously known about how the carboxymethylated DNA lesions perturb DNA replication in human cells. Here, we investigated the effects of five carboxymethylated DNA lesions, i.e. O6-CMdG, N6-CMdA, N4-CMdC, N3-CMdT and O4-CMdT on the efficiency and fidelity of DNA replication in HEK293T human embryonic kidney cells. We found that, while neither N6-CMdA nor N4-CMdC blocked DNA replication or induced mutations, N3-CMdT, O4-CMdT and O6-CMdG moderately blocked DNA replication and induced substantial frequencies of T→A (81%), T→C (68%) and G→A (6.4%) mutations, respectively. In addition, our results revealed that CRISPR-Cas9-mediated depletion of Pol η resulted in significant drops in bypass efficiencies of N4-CMdC and N3-CMdT. Diminution in bypass efficiencies was also observed for N6-CMdA and O6-CMdG upon depletion of Pol κ, and for O6-CMdG upon removal of Pol ζ. Together, our study provided molecular-level insights into the impacts of the carboxymethylated DNA lesions on DNA replication in human cells, revealed the roles of individual translesion synthesis DNA polymerases in bypassing these lesions, and suggested the contributions of O6-CMdG, N3-CMdT and O4-CMdT to the mutations found in p53 gene of human gastrointestinal cancers.

Biological Evaluation of DNA Biomarkers in a Chemically Defined and Site-Specific Manner

“…O 4 -carboxymethylthymidine ( O 4 -CMdT) is a strong replication block (51%) and very mutagenic, causing 86% T>C mutation [36]. N 3 -CMdT, O 4 -CMdT and O 6 -carboxymethyl-dG ( O 6 -CMdG) moderately block DNA replication in human cells [48]. N 3 -CMdT causes T>A (81%) mutation; O 4 -CMdT causes T>C (68%) mutation; O 6 -CMdG causes G>A (6.4%) mutation; neither N 6 -CMdA nor N 4 -CMdC block replication or induce mutation [48].…”

High Sensitivity of Human Translesion DNA Synthesis Polymerase κ to Variation in O6-Carboxymethylguanine Structures

“…25 Therefore, the data suggested an involvement of TLS Pols κ and ζ in error-free bypass of O 6 -CMG whereas Pol ι seems to be responsible for the mutagenic bypass of the adduct. 25 In line with these findings, primer extension experiments demonstrated that the replicative Pol δ was only moderately blocked. Additionally, Pol κ, which only inserted dCTP, was the highest fidelity Pol amongst the TLS Pols tested, 26 and Pols η and ι frequently misincorporated dATP and dTTP opposite O 6 -CMG.…”

“…In HEK293 cells transfected with plasmids containing a site-specific O 6 -CMG, the adduct was a modest block for DNA synthesis and was moderately mutagenic. 25 Adduct bypass decreased in cells deficient in Pol κ and ζ and O 6 -CMG induced exclusively GCAT mutations at a frequency of 6.4% when Pol ι was genetically depleted. 25 Therefore, the data suggested an involvement of TLS Pols κ and ζ in error-free bypass of O 6 -CMG whereas Pol ι seems to be responsible for the mutagenic bypass of the adduct.…”

“…25 Adduct bypass decreased in cells deficient in Pol κ and ζ and O 6 -CMG induced exclusively GCAT mutations at a frequency of 6.4% when Pol ι was genetically depleted. 25 Therefore, the data suggested an involvement of TLS Pols κ and ζ in error-free bypass of O 6 -CMG whereas Pol ι seems to be responsible for the mutagenic bypass of the adduct. 25 In line with these findings, primer extension experiments demonstrated that the replicative Pol δ was only moderately blocked.…”

“…Overall, the data from both isolated enzymes and cells suggest the miscoding potential of O 6 -CMG depends on which Pol is functional in the TLS bypass of O 6 -CMG and its mechanism of processing the adduct. 25,26 Figure 1. O 6 -CMG modifications used for the SAR study.…”