FIG. 3.

Inhibition of MMR reaction by DNA-containing carcinogen adducts. G-T mismatch repair was performed as described previously (30) by using 50 μg of HeLa nuclear extracts and 100 ng of a heteroduplex containing a single G-T mismatch. Repair assays were performed in reaction mixtures containing 100 ng of the G-T mismatched heteroduplex, the indicated competitor DNA, and 50 μg of HeLa nuclear extracts. The reaction mixtures were incubated at 37°C for 15 min. After phenol and ether extraction, DNA substrates were recovered by ethanol precipitation and digested with restriction endonucleases to score the repair of the G-T heteroduplex. For the homoduplex (G-C) or carcinogen-modified homoduplexes (G-C/B[a]PDE, and G-C/AAAF), restriction endonucleases HindIII and Bsp106 were used to detect the repair of the G-T substrate, producing 3.1- and 3.3-kb fragments (see Fig. 1). For the M13-fd heteroduplex, HindIII and BseRI (producing 2.8- and 3.6-kb fragments) were used since Bsp106 cleaves the M13-fd substrate into fragments of the same size as those obtained by HindIII and Bsp106 cleavage of the repaired G-T substrate.