Carcinogenesis, Vol 18, 1407-1413, Copyright © 1997 by Oxford University Press
MN Routledge, JM Allan and RC Garner
To investigate the use of UvrB-binding to detect DNA damage, mobility shift
gel electrophoresis was used to detect binding of UvrB protein to a 136 bp
DNA fragment that was randomly adducted with aflatoxin B1 8,9- epoxide and
end-labelled with 32P. After polyacrylamide gel electrophoresis, the
shifted band that contained DNA bound by UvrB was quantified as a
percentage of total radioactive substrate DNA. This method was applied to
analyse plasmid DNA that was adducted with various DNA modifying agents in
vitro. These adducts competed for UvrB- binding to the labelled substrate.
By competing for UvrB-binding with 10 ng of plasmid DNA that was adducted
with known levels of aflatoxin B1, 2-amino-3-methylimidazo[4,5-f]quinoline,
or benzo[a]pyrene diol epoxide, UvrB competition could be quantified for
DNA adducted with between one adduct in 10(2) and one adduct in 10(5)
normal nucleotides. However, plasmid DNA exposed to N-methyl-N-nitrosourea
or methylene blue + visible light, did not compete for UvrB-binding, even
though the presence of UvrABC sensitive sites were confirmed on this DNA by
a UvrABC incision assay. Mono-adducted 96-bp DNA substrates, which
contained an internal 32P-label and either a single apurinic site,
aflatoxin B1-guanine adduct, O6-methylguanine, 8-oxo-deoxyguanosine or
non-adducted guanine, were also used as substrates for UvrA- and UvrB-
binding to examine the stability of UvrB-DNA complexes with specific
adducts. Under similar conditions used for the competition assay,
significant UvrB-binding was seen only for the aflatoxin adducted
substrate. These results suggest that stability of UvrB-binding varies
greatly between bulky and non-bulky adducts. It was also found that rat
liver DNA from untreated rats inhibited UvrB-binding to the substrate DNA
in the competition assay, to a degree that was equivalent to competition
with plasmid adducted at one adduct in 10(3) normal nucleotides.
ARTICLES
Detection of DNA damage by Escherichia coli UvrB-binding competition assay is limited by the stability of the UvrB-DNA complex
Biology Department, University of York, Heslington, UK.
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