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Schorr, Stephanie (2010): Replication of Bulky DNA Adducts: Replikation von sterisch anspruchsvollen DNA Addukten. Dissertation, LMU München: Faculty of Chemistry and Pharmacy
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Abstract

Aromatic amines are known to be strong carcinogens. After metabolic activation, they react as electrophilic arylnitrenium ions with nucleophilic functionalities of the DNA duplex interfering and disrupting DNA and RNA synthesis and leading to mutations. Preferred reaction sites are the amino groups of adenine and guanine and particularly the C8-position of guanine. During metabolic activation, aromatic amines are enzymatically acetylated at N8 position. The non-acetylated lesions reduce the replication efficiency, but are in general faithfully bypassed by high fidelity polymerases. In contrast, the acetylated derivatives block replicative polymerases but can be bypassed with special low-fidelity polymerases. The translesion synthesis DNA polymerase η, for instance, is able to bypass C8 bulky adduct lesions such as the widely studied 2-aminofluorene-dG (AF-dG) and its acetylated analogue (AAF-dG) mainly in an error-free manner. The distinct mutagenic properties of the acetylated and non-acetylated aromatic amine lesions are presumably caused by their different conformational preferences. While the non-acetylated lesions exist in both syn and anti conformation, the corresponding acetylated lesion seems to adopt the syn-conformation with high preference. The mechanism that allows low-fidelity polymerases such as Pol η to replicate past acetylated AAF-dG lesions is still unknown. In this thesis work, the mechanism of the error-free bypass of acetylated aromatic amine dG adducts such as the acetylaminofluorene-dG (AAF-dG) by Pol η is investigated. For that reason, AAF-dG as well as the guanine adducts of other aromatic amines (aniline, 2-aminonaphthalene, 2-aminoanthracene and 1-aminopyrene) were synthesized and successfully incorporated into various oligonucleotides via automated solid-phase DNA synthesis. The synthesized bulky adduct containing oligonucleotides were used for crystallization and for primer extension studies with the translesion synthesis (TLS) polymerase η from S. cerevisiae in order to get insights into the bypass mechanism of bulky adducts by this enzyme. In the present work, structural evidence is provided that yeast Pol η bypasses the bulky adducts AAF-dG and 2-acetylaminoanthracene-dG (AAA-dG) by rotation of the DNA around the bulky moiety, while keeping the AAF-dG in syn conformation.