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 shiftgel electrophoresis was used to detect binding of UvrB protein to a 136 bpDNA fragment that was randomly adducted with aflatoxin B1 8,9- epoxide andend-labelled with 32P. After polyacrylamide gel electrophoresis, theshifted band that contained DNA bound by UvrB was quantified as apercentage of total radioactive substrate DNA. This method was applied toanalyse plasmid DNA that was adducted with various DNA modifying agents invitro. These adducts competed for UvrB- binding to the labelled substrate.By competing for UvrB-binding with 10 ng of plasmid DNA that was adductedwith known levels of aflatoxin B1, 2-amino-3-methylimidazo[4,5-f]quinoline,or benzo[a]pyrene diol epoxide, UvrB competition could be quantified forDNA adducted with between one adduct in 10(2) and one adduct in 10(5)normal nucleotides. However, plasmid DNA exposed to N-methyl-N-nitrosoureaor methylene blue + visible light, did not compete for UvrB-binding, eventhough the presence of UvrABC sensitive sites were confirmed on this DNA bya UvrABC incision assay. Mono-adducted 96-bp DNA substrates, whichcontained an internal 32P-label and either a single apurinic site,aflatoxin B1-guanine adduct, O6-methylguanine, 8-oxo-deoxyguanosine ornon-adducted guanine, were also used as substrates for UvrA- and UvrB-binding to examine the stability of UvrB-DNA complexes with specificadducts. Under similar conditions used for the competition assay,significant UvrB-binding was seen only for the aflatoxin adductedsubstrate. These results suggest that stability of UvrB-binding variesgreatly between bulky and non-bulky adducts. It was also found that ratliver DNA from untreated rats inhibited UvrB-binding to the substrate DNAin the competition assay, to a degree that was equivalent to competitionwith plasmid adducted at one adduct in 10(3) normal nucleotides.