posted on 2020-03-31, 21:19authored byClaudia
M. N. Aloisi, Arman Nilforoushan, Nathalie Ziegler, Shana J. Sturla
DNA mutations can result from replication
errors due to different
forms of DNA damage, including low-abundance DNA adducts induced by
reactions with electrophiles. The lack of strategies to measure DNA
adducts within genomic loci, however, limits our understanding of
chemical mutagenesis. The use of artificial nucleotides incorporated
opposite DNA adducts by engineered DNA polymerases offers a potential
basis for site-specific detection of DNA adducts, but the availability
of effective artificial nucleotides that insert opposite DNA adducts
is extremely limited, and furthermore, there has been no report of
a quantitative strategy for determining how much DNA alkylation occurs
in a sequence of interest. In this work, we synthesized an artificial
nucleotide triphosphate that is selectively inserted opposite O6-carboxymethyl-guanine DNA by an engineered
polymerase and is required for DNA synthesis past the adduct. We characterized
the mechanism of this enzymatic process and demonstrated that the
artificial nucleotide is a marker for the presence and location in
the genome of O6-carboxymethyl-guanine.
Finally, we established a mass spectrometric method for quantifying
the incorporated artificial nucleotide and obtained a linear relationship
with the amount of O6-carboxymethyl-guanine
in the target sequence. In this work, we present a strategy to identify,
locate, and quantify a mutagenic DNA adduct, advancing tools for linking
DNA alkylation to mutagenesis and for detecting DNA adducts in genes
as potential diagnostic biomarkers for cancer prevention.