DinB, a Y-family DNA polymerase, is conserved among all domains of life; however, its endogenous substrates have not been identified. DinB is known to synthesize accurately across a number of N 2 -dG lesions. Methylglyoxal (MG) is a common byproduct of the ubiquitous glycolysis pathway and induces the formation of N 2 -(1-carboxyethyl)-2-deoxyguanosine (N 2 -CEdG) as the major stable DNA adduct. Here, we found that N 2 -CEdG could be detected at a frequency of one lesion per 10 7 nucleosides in WM-266-4 human melanoma cells, and treatment of these cells with MG or glucose led to a dose-responsive increase in N 2 -CEdG formation. We further constructed single-stranded M13 shuttle vectors harboring individual diastereomers of N 2 -CEdG at a specific site and assessed the cytotoxic and mutagenic properties of the lesion in wild-type and bypass polymerase-deficient Escherichia coli cells. Our results revealed that N 2 -CEdG is weakly mutagenic, and DinB (i.e., polymerase IV) is the major DNA polymerase responsible for bypassing the lesion in vivo. Moreover, steady-state kinetic measurements showed that nucleotide insertion, catalyzed by E. coli pol IV or its human counterpart (i.e., polymerase ), opposite the N 2 -CEdG is both accurate and efficient. Taken together, our data support that N 2 -CEdG, a minor-groove DNA adduct arising from MG, is an important endogenous substrate for DinB DNA polymerase.glycolysis ͉ mutagenesis ͉ polymerase ͉ translesion synthesis

Reactive oxygen species (ROS) can be induced by both endogenous and exogenous processes and they can damage biological molecules including nucleic acids. It was shown that X-or γ-ray irradiation of aqueous solutions of DNA, during which · OH is one of the major ROS, can lead to the formation of intrastrand crosslink lesions where the neighboring nucleobases in the same DNA strand are covalently bonded. Previous 32 P-postlabeling studies suggested that the intrastrand crosslink lesions may arise from Fenton reaction, which also induces the formation of · OH; the structures of the proposed intrastrand crosslink lesions, however, have not been determined. Here we showed for the first time that the treatment of calf thymus DNA with Cu(II)/H 2 O 2 /ascorbate could lead to the formation of an intrastrand crosslink lesion, i.e., G^T, where the C8 of guanine is covalently bonded to the neighboring 3′ thymine through its methyl carbon. LC-MS/MS quantification results showed dose-responsive formation of G^T. In addition, the yield of the intrastrand crosslink was approximately three orders of magnitude lower than those of commonly observed single-base lesions, that is, 8-oxo-7,8-dihydro-2′-deoxyguanosine, 5-(hydroxymethyl)-2′-deoxyuridine and 5-formyl-2′-deoxyuridine. The induction of intrastrand crosslink lesion in calf thymus DNA by Fenton reagents in vitro suggests that this type of lesion might be formed endogenously in mammalian cells.

Reactive oxygen species (ROS) can be induced by both endogenous and exogenous processes, and they can damage biological molecules including nucleic acids. Exposure of isolated DNA to X/γ-rays and Fenton reagents was shown to lead to the formation of intrastrand cross-link lesions where the neighboring nucleobases in the same DNA strand are covalently bonded. By employing HPLC coupled with tandem mass spectrometry (LC-MS/MS) with the isotope dilution method, we assessed quantitatively the formation of a guanine–cytosine (G[8-5]C) intrastrand cross-link lesion in HeLa-S3 cells upon exposure to γ-rays. The yield of the G[8-5]C cross-link was 0.037 lesions per 109 nucleosides per Gy, which was ∼300 times lower than that of 5-formyl-2′-deoxyuridine (0.011 lesions per 106 nucleosides per Gy) under identical exposure conditions. We further constructed a single-stranded M13 genome harboring a site-specifically incorporated G[8-5]C lesion and developed a novel mass spectrometry-based method for interrogating the products emanating from the replication of the genome in Escherichia coli cells. The results demonstrated that G[8-5]C blocked considerably DNA replication as represented by a 20% bypass efficiency, and the lesion was significantly mutagenic in vivo, which included a 8.7% G→T and a 1.2% G→C transversion mutations. DNA replication in E. coli hosts deficient in SOS-induced polymerases revealed that polymerase V was responsible for the error-prone translesion synthesis in vivo.

Human cells are constantly exposed to environmental and endogenous agents which can induce damage to DNA. Understanding the implications of these DNA modifications in the etiology of human diseases requires the examination about how these DNA lesions block DNA replication and induce mutations in cells. All previously reported shuttle vector-based methods for investigating the cytotoxic and mutagenic properties of DNA lesions in cells have low-throughput, where plasmids containing individual lesions are transfected into cells one lesion at a time and the products from the replication of individual lesions are analyzed separately. The advent of next-generation sequencing (NGS) technology has facilitated investigators to design scientific approaches that were previously not technically feasible or affordable. In this study, we developed a new method employing NGS, together with shuttle vector technology, to have a multiplexed and quantitative assessment of how DNA lesions perturb the efficiency and accuracy of DNA replication in cells. By using this method, we examined the replication of four carboxymethylated DNA lesions and two oxidatively induced bulky DNA lesions including (5′S) diastereomers of 8,5′-cyclo-2′-deoxyguanosine (cyclo-dG) and 8,5′-cyclo-2′-deoxyadenosine (cyclo-dA) in five different strains of Escherichia coli cells. We further validated the results obtained from NGS using previously established methods. Taken together, the newly developed method provided a high-throughput and readily affordable method for assessing quantitatively how DNA lesions compromise the efficiency and fidelity of DNA replication in cells.

G[8-5m]T, a guanine-thymine intrastrand cross-link lesion where the C8 of guanine is covalently bonded to the neighboring 3'-thymine through its methyl carbon, was previously shown to form in an aqueous solution of duplex DNA upon exposure to gamma- or X-rays and in calf thymus DNA treated with Fenton reagents. Here, we employed LC-MS/MS and demonstrated for the first time that this lesion could be induced in a dose-dependent manner in human Hela-S3 cells upon exposure to gamma-rays. We further carried out in vitro replication studies on a substrate containing a site-specifically incorporated G[8-5m]T, and our results showed that the Klenow fragment of Escherichia coli DNA polymerase I stopped synthesis mostly after incorporating the correct nucleotide dAMP opposite the 3'-thymine moiety of the lesion. On the other hand, yeast Saccharomyces cerevisiae DNA polymerase eta (pol eta) was able to replicate past the cross-link lesion, but with markedly reduced efficiency in nucleotide incorporation opposite the 5'-guanine of the lesion. Steady-state kinetic analyses for nucleotide incorporation by yeast pol eta showed that the 5'-guanine portion of the lesion also decreased pronouncedly the fidelity of nucleotide incorporation; the insertion of dAMP and dGMP was favored over that of the correct nucleotide, dCMP. The above results support the conclusion that oxidative intrastrand cross-link lesions, if not repaired, can be cytotoxic and mutagenic.