Replication of Bulky DNA Adducts

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.