Archive | 2019
Gene-targeted identification of DNA lesions to unravel mechanisms of mutagenesis
Abstract
3 Abstract The integrity of our genomic material is continuously threatened by exposure to endogenous and exogenous DNA-damaging chemicals. The resulting chemical adducts to DNA can initiate adverse biological consequences, including cell death and DNA mutations that potentially lead to cancer. Our capacity to relate mutagenesis with specific DNA adducts is limited by the lack of strategies to measure the early event of DNA damage within genomic loci. Furthermore, inadequate knowledge regarding the cellular processes that handle the DNA adducts hinders our understanding of mutagenicity-driving factors in response to DNA damage in cells. The work described in this thesis concerns addressing both the gap in technology and the gap in knowledge with regard to a type of mutagenic, biologically relevant DNA adducts. The main achievements of this work were the development of strategies for the detection of the DNA adducts in a target DNA sequence, and the identification of cellular mechanisms that alleviate the mutagenicity induced by the DNA adducts. In Chapter 1, topics further discussed in this thesis are introduced. DNA adduct-directed artificial nucleotides have been developed as a basis for interrogating damaged DNA in duplex hybridization or polymerase-mediated synthesis contexts. Strategies based on artificial nucleotides are employed for the work of Chapters 2 and 3. An overview of the studies presented in this thesis is given at the end of the chapter. In Chapter 2, a strategy to detect the mutagenic O6-methylguanine DNA adduct in the DNA sequence of a cancer-relevant gene is developed. A DNA primer containing an artificial nucleoside analogue that stabilizes O6-methylguanine in the duplex allowed replication of DNA by an engineered DNA polymerase only when the artificial nucleotide was paired with O6-methylguanine. The artificial nucleotide-modified primer effectively marked presence and position of O6-methylguanine in the sequence. Furthermore, thanks to high specificity of replication, the modified DNA primer could be extended and amplified only when O6-methylguanine damaged DNA was present, and signal was obtained that linearly increased with the amount of O6-methylguanine in the sample. The presented strategy represents the first instance of polymerase amplification-based O6-methylguanine detection to single base resolution. This approach could be used to detect O6-methylguanine in other cancerrelevant gene sequences. In Chapter 3, the scope and performance of artificial nucleotide-based detection of DNA adducts reported in the previous chapter is expanded by the introduction of an analytical measurement that allows for the quantification of the DNA adducts. A heterocyclic imidic nucleotide triphosphate analogue is synthesized for the detection and quantification of the mutagenic O6carboxymethylguanine (O6-CMG), which has been associated to cancer development linked to meat consumption. The nucleotide analogue was incorporated selectively opposite O6-CMG over undamaged G by an engineered DNA polymerase, and was required for replication of DNA past O6-