Detection of DNA damage by Escherichia coli UvrB-binding competition assay is limited by the stability of the UvrB-DNA complex.
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.
Routledge
M N
MN
Biology Department, University of York, Heslington, UK.
Allan
J M
JM
Garner
R C
RC
D016604:Aflatoxin B1 / Q000378:metabolism; D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism*; D001667:Binding, Competitive; D004247:DNA / Q000378:metabolism*; D018736:DNA Adducts / Q000378:metabolism; D004249:DNA Damage; D004265:DNA Helicases; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D006801:Humans; D051381:Rats
D001426:Bacterial Proteins; D018736:DNA Adducts; D029968:Escherichia coli Proteins; C058093:UvrB protein, E coli; C072611:aflatoxin B1-DNA adduct; D004247:DNA; D016604:Aflatoxin B1; D004265:DNA Helicases