Kinetics of deamination and Cu(II)/H2O2/Ascorbate-induced formation of 5-methylcytosine glycol at CpG sites in duplex DNA

Mutation in p53 tumor suppressor gene is a hallmark of human cancers. Six major mutational hotspots in p53 contain methylated CpG (mCpG) sites, and C →T transition is the most common mutation at these sites. It was hypothesized that the formation of 5-methylcytosine glycol induced by reactive oxygen species, its spontaneous deamination to thymine glycol and the miscoding property of the latter may account, in part, for the ubiquitous C →T mutation at CpG site. Here, we assessed the kinetics of deamination for two diastereomers of 5-methylcytosine glycol in duplex DNA. Our results revealed that the half-lives for the deamination of the (5S,6S) and (5R,6R) diastereomers of 5-methylcytosine glycol in duplex DNA at 37°C were 37.4 ± 1.6 and 27.4 ± 1.0 h, respectively. The deamination rates were only slightly lower than those for the two diastereomers in mononucleosides. Next, we assessed the formation of 5-methyl-2′-deoxycytidine glycol in the form of its deaminated product, namely, thymidine glycol (Tg), in methyl-CpG-bearing duplex DNA treated with Cu(II)/H2O2/ascorbate. LC-MS/MS quantification results showed that the yield of Tg is similar as that of 5-(hydroxymethyl)-2′-deoxycytidine. Together, our data support that the formation and deamination of 5-methylcytosine glycol may contribute significantly to the C →T transition mutation at mCpG dinucleotide site

High-throughput analysis of the mutagenic and cytotoxic properties of DNA lesions by next-generation sequencing

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

Formation and genotoxicity of a guanine–cytosine intrastrand cross-link lesion in vivo

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

Probe Oxidative Damage in DNA Charge Transfer Process

As a hydrophilic biopolymer, a DNA molecule is surrounded by water molecules in aqueous solution. The charge hopping mechanism indicates the competition between radical cation quenching by water molecules and migration along DNA partially determines the distance and efficiency of charge transport in DNA. Lipid can effectively bind DNA to induce hydrophobic environment around the DNA helix and reduce the water contact with bases in the DNA duplex. Therefore, the effect of water molecules on charge transport can be studied by comparison between nature DNA and DNA-lipid complexes. We synthesized several cationic lipids with various lengths of dialkyl chain (2, 8, 18) and spermine (Sp4+) binding core in this research, which posses strong DNA binding affinity due to their multi-charged spermine head-groups. Among those, C8GlySp4+ and C2GlySp4+ can form stable complex with DNA oligomer in aqueous solution, characterized by time dependent UV and CD spectrometry. C2GlySp4+ showed the similar inhibition on oxidative damage in GG steps as spermine while C8GlySp4+ demonstrated much more significant prohibitive effect at the same concentration. Since all the lipids bear the same binding core, they should afford the similar binding affinity towards DNA duplexes. we attributed the observation to the longer length of dialkyl group in C8GlySp4+, which can more effectively shield the DNA duplex from the water molecules than either spermine or C2GlySp4+. A kinetic model based on phonon-assist polaron hopping mechanism was proposed to rationalize the experimental results. The finding may give insight on the protection of DNA oxidative damage by reducing the access of the water molecule to DNA duplex and may have potential impact on the application of DNA as conducting biopolymer and protection of DNA in biological system.Ph.D.Committee Chair: Gary B Schuster; Committee Member: Andrew Lyon; Committee Member: Nicolas V Hud; Committee Member: Suzanne B Shuker; Committee Member: Uzi Landma

Part I Charge transfer in DNA containing modified guanine bases: Steric and engergetic control of reactivity : Part II Synthesis of DNA binding lipids to induce hydrophobic environment

M.S.Gary B. Schuste

and CpG-methylated DNA induced by Fenton-type reagents

intrastrand cross-link lesions in unmethylate