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 t o a 136 bp DNA fragment that was randomly adducted with aflatoxin B-1 8,9-epoxide and end-labelled with P-32, After polyacrylamide gel elect rophoresis, the shifted band that contained DNA bound by UvrB was quan tified 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 p lasmid DNA that was adducted with known levels of aflatoxin B-1, 2-ami no-3-methylimidazo [4,5-f]quinoline, or benzo[alpha]pyrene diol epoxid e, UvrB competition could be quantified for DNA adducted with between one adduct in 10(2) and one adduct in 10(5) normal nucleotides. Howeve r, plasmid DNA exposed to N-methyl-N-nitrosourea or methylene blue + v isible light, did not compete for UvrB-binding, even though the presen ce of UvrABC sensitive sites were confirmed on this DNA by a UvrABC in cision assay, Mono-adducted 96-bp DNA substrates, which contained an i nternal P-32-label and either a single apurinic site, aflatoxin B-1-gu anine adduct, O-6-methylguanine, 8-oxodeoxyguanosine or non-adducted g uanine, were also used as substrates for UvrA- and UvrB-binding to exa mine the stability of UvrB-DNA complexes with specific adducts, Under similar conditions used for the competition assay, significant UvrB-bi nding was seen only for the aflatoxin adducted substrate, These result s suggest that stability of UvrB-binding varies greatly between bulky and non-bulky adducts, It was also found that rat liver DNA from untre ated rats inhibited UvrB-binding to the substrate DNA in the competiti on assay, to a degree that was equivalent to competition with plasmid adducted at one adduct in 10(3) normal nucleotides.