Chemical Biology of DNA damage

The Exposome

While disease risk is often attributed to genetics, environmental factors represent a much larger contribution to risk of disease development. The exposome, a term first used in 2005 to describe the life-course environmental exposures, is discussed in three categories: general external exposures, specific external exposures, and internal exposures. General external exposures include environmental factors such as urban-rural environment and socioeconomic status. Specific external exposures include lifestyle choices such as occupation or tobacco and alcohol use. Internal exposures come from processes such as metabolism. Each of these three categories of exposure can lead to the formation carcinogen-DNA adducts.

Figure 1: The exposome

Formation of DNA adducts upon exposure to carcinogens

Genomic DNA is exposed to various exogenous and endogenous agents, including bis-electrophiles, α,β, unsaturated carbonyls, ionizing radiation and free radicals. This exposure results in DNA damage such as alkylation of nucleobases, apurinic site formation, DNA strand breaks, trapping of cellular proteins on genomic DNA to form super bulky DNA-Protein Cross-links (DPCs), DNA-DNA crosslinks, etc. While most of these lesions will be repaired by various DNA repair machineries, there are lesions/DNA adducts which are very persistent and may potentially contribute to mutagenesis and carcinogenesis.

Figure 2: Examples of DNA adducts that form when DNA is exposed to exogenous and endogenous agents

Formamidopyriminidine adducts of 1,3-butadiene

One of our many projects revolves around the synthesis, structural characterization and detection of formamidopyrimidine (FAPy) adducts derived from epoxybutene and diepoxybutane in cells. Our lab recently reported the first successful synthesis of EB-FAPy-dG and its detection in human cells. We have also established the genotoxicity and mutagenicity of this lesion. We then additionally developed a small-scale synthesis and sensitive isotope-dilution nanoLC-ESI+ -HRMS/MS methodology for the detection and quantitation of DEB-FAPy-dG. Further total synthesis of the DEB-FAPy-phosphoramidite is underway to incorporate these lesions into DNA and study their effects on cell cycle.

Figure 3. Synthesis of EB-FAPy.

Solid phase synthesis of DNA and in vitro DNA polymerase bypass assays

To study the effects of DNA adducts on DNA replication, we utilize solid-phase DNA synthesis. The DNA adducts we synthesize in our lab are further converted into their respective phosphoramidites and incorporated into DNA oligonucleotides.

These DNA adduct containing oligonucleotides can then be utilized for in vitro DNA replication experiments. These experiments evaluate the ability of DNA polymerases to bypass the DNA lesion and the frequency of errors in DNA replication due to the presence of the DNA adducts.

Further work on this project will aim to introduce DNA adduct containing plasmids into cells to study these adducts in a cellular context and study their downstream effects.

Figure 4. Solid-phase synthesis of DNA oligonucleotides

Figure 5. In vitro DNA replication experiments

Representative Publications:

Groehler IV, A., et al. N6-(2-Deoxy-D-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-(2-hydroxy-3-buten-1-yl)-formamidopyrimidine Adducts of 1,3-Butadiene: Synthesis, Structural Identification, and Detection in Human Cells. Chemical Research in Toxicology 2018, 31 (9), 885–897. Article
Chesner, L. N., et al. Cellular Repair of DNA–DNA Cross-Links Induced by 1,2,3,4-Diepoxybutane. International Journal of Molecular Sciences 2017, 18 (5). Article
Chang, S. C., et al. 1,3-Butadiene-Induced Adenine DNA Adducts Are Genotoxic but Only Weakly Mutagenic When Replicated in Escherichia Coli of Various Repair and Replication Backgrounds. Chemical Research in Toxicology 2017, 30 (5), 1230–1239. Article.

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