Quantification and Sequencing of Oxidative DNA Damage
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Date
2024
Publication Type
Doctoral Thesis
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Abstract
DNA oxidation occurs frequently in our cells and is linked to both beneficial and detrimental outcomes concerning human health. Exposure to chemicals or radiation can promote the production of reactive oxygen species (ROS), which oxidize DNA to predominantly form 8-oxoguanine (8-oxoG). 8-oxoG is repaired through base excision repair (BER), which employs enzymes such as 8-oxoG glycosylase 1 (Ogg1) to safeguard the DNA from genomic instability. However, incomplete or deficient repair of 8-oxoG can result in mutations or cell death, contributing to carcinogenesis and aging. Thus, understanding how exposure to chemicals or radiation produce 8-oxoG and how it is repaired is important to assess its impact on human health. Current methods to characterize 8-oxoG are limited in specificity and resolution, causing inconsistent results, and preventing comprehensive elucidation of its distribution in the human genome. Furthermore, 8-oxoG analyses are challenged by artifactual DNA oxidation and strategies in reducing artifacts often remain unvalidated. The goals of this thesis are to develop methods to quantify and sequence DNA damage as tools for studying how exposure to chemicals and radiation promote the formation of 8-oxoG in the human genome and explore the impact of enzymatic repair on 8-oxoG levels and distribution. Two methods, fluoroclick and click-code-seq, were optimized for the quantification of chemical- and radiation-induced DNA damage and sequencing of endogenous 8-oxoG in human cells.
Chapter 1 outlines oxidative stress and its role in causing DNA oxidation, highlighting its significance for human health. Various forms of oxidative DNA damage are presented with a special focus on the major DNA oxidation product 8-oxoG. This introduction describes the repair mechanisms associated with 8-oxoG and its impact, both harmful and beneficial, in cells. Finally, current techniques used to measure and sequence 8-oxoG are described, including the challenges involved in accurately detecting 8-oxoG in the human genome.
Chapter 2 is focused on the development of fluoroclick, a method to quantify oxidative DNA damage, apurinic/apyrimidinic (AP) sites, and single-strand breaks (SSBs) in DNA. Fluoroclick is based on using genomic repair enzymes to produce 3'-OH-containing gaps at damage sites, into which propargyl-modified nucleotides are inserted to serve as a reactive handle for covalent linkage with a fluorophore via copper-catalyzed azide-alkyne cycloaddition (CuAAC). Using oligonucleotides with a single 8-oxoG to establish suitable reaction conditions for fluoroclick, we found that AF594 fluorescence of fluoroclick-modified DNA correlated with 8-oxoG concentration. After adaptation to quantify damage in human gDNA, fluoroclick was used to optimize click-code-seq for accurate 8-oxoG sequencing. By mapping endogenous 8-oxoG in human chronic-myeloid-leukemia-derived HAP1 cells we observed a correlation with oxidative stress-related mutational signatures. As a result of this study, fluoroclick and click-code-seq are available as complementary tools to precisely quantify and sequence 8-oxoG in the human genome. These tools can contribute to an improved understanding of the factors that influence the distribution of 8-oxoG in the human genome.
Chapter 3 is centered on the formation and repair of 8-oxoG in response to UVA and potassium bromate (KBrO3) exposure of human epithelial osteosarcoma-derived U2OS cells. To address the influence of repair on 8-oxoG levels, we exposed U2OS wildtype (WT) and Ogg1-deficient cells to 10 J/cm2 UVA or 50 mM KBrO3 followed by recovery up to 24 h. Levels of 8-oxoG, AP sites and SSBs were quantified using fluoroclick. We observed a significant 8-oxoG increase in KBrO3-exposed compared to unexposed cells, which fully declined after 24 h in WT but not in Ogg1-deficient cells. We attributed this to ongoing repair and observed a similar decline in UVA-exposed WT cells. These data serve as a basis for further sequencing analyses and will eventually contribute to understand how 8-oxoG profiles are shaped by exposure to chemicals or radiation and the impact of cellular repair mechanisms.
Chapter 4 is a summary of the results presented in thesis, specifically the development of fluoroclick and click-code-seq and their application to characterize 8-oxoG in the human genome. Methodology and data are compared with published results from other research groups with a focus on current limitations and potential solutions. Finally, an outlook for future research is presented.
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Examiner: Sturla, Shana J.
Examiner : Beer, Hans-Dietmar
Examiner : Poetsch, Anna
Examiner: Takhaveev, Vakil
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ETH Zurich
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Subject
Oxidative DNA Damage, DNA Damage Sequencing, 8-oxoG
Organisational unit
03853 - Sturla, Shana / Sturla, Shana