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.

Figure 1.

Formation of diazoacetate and its induction of carboxymethylated DNA lesions.

Figure 2.

Schematic diagrams showing the procedures for the preparation of the lesion-bearing plasmid (A) and the SSPCR-CRAB assay (B). ‘X’ indicates the carboxymethylated lesions. The C/C mismatch site is underlined. ‘P1’ represents one of primers for PCR and contains a G as the terminal 3΄-nucleotide corresponding to the C/C mismatch site of the lesion-bearing genome. It also contains a C/A mismatch three bases away from its 3΄-end for improving PCR specificity. ‘M’ designates the nucleobase incorporated at the lesion site after replication, and ‘N’ represents the paired nucleobase of ‘M’ in the complementary strand.

Figure 3.

Restriction digestion and post-labeling method for determining the bypass efficiencies and mutation frequencies of the carboxymethylated lesions in HEK293T cells. (A) Sample processing for restriction digestion using NcoI and SfaNI and post-labeling assay (p* indicates a ³²P-labeled phosphate group). The recognition sequences for restriction enzymes are designated with arrows. (B) Representative gel images showing the NcoI/SfaNI-produced restriction fragments of interest. The restriction fragment arising from the competitor vector, i.e. d(CATGGCGATATGCTGT), is designated as ‘16 mer’; ‘13 mer C’, ‘13 mer A’, ‘13 mer G’ and ‘13 mer T’ represent the standard synthetic ODNs d(CATGGCGNGCTGT), where ‘N’ is C, A, G and T, respectively.

Figure 4.

Restriction digestion followed by LC–MS/MS for the identification of restriction digestion products (with NcoI and SfaNI) of PCR products of progeny genome arising from the replication of N3-CMdT and O⁴-CMdT lesions in HEK293T cells. Shown are the MS/MS for monitoring the fragmentations of the [M–3H]^3– ions of 5΄-AATTACAGCTCGC-3΄ (with T→A mutation, A) and 5΄-AATTACAGCGCGC-3΄ (with T→C mutation, B). The restriction digestion method was the same as what was described in Figure 2B. Shown in the insets are schemes summarizing the observed fragment ions and higher-resolution ‘ultra-zoom scan’ ESI-MS for monitoring the [M–3H]^3- ions of the T→A and T→C mutation products.

Figure 5.

Relative bypass efficiencies (A) and mutation frequencies (B) of the carboxymethylated DNA lesions. The data represent the mean and S.E.M. of results from three independent cellular replication experiments. The P values referred to the differences between the wild-type (WT) HEK293T cells and the relevant polymerase-deficient cells, and they were calculated using two-tailed, unpaired t-test. *P < 0.05; ***P < 0.001.

Figure 6.

Proposed base-pairings involved in correct nucleotide incorporations opposite N⁶-CMdA and N⁴-CMdC, along with the incorrect nucleotide insertions opposite other carboxymethylated DNA lesions.