Mutation Spectrum Induced by 8-Bromoguanine, a Base Damaged by Reactive Brominating Species, in Human Cells

Figure 4

Excision of adenine mispaired with 8-bromoguanine (8BrG) by MUTYH protein. (a) Time-course assay for the cleavage of 30-mer double-stranded oligonucleotides containing an A:8BrG or an A:8oxoG mispair or not containing a mispair (A:T) by MUTYH protein. The protein was incubated at 37°C for 0–120 min with each oligonucleotide. The amount of cleavage products as a proportion of the total oligonucleotides was calculated as the % incision. The % incision values were shown as the mean ± standard deviation of data from three independent experiments. The lower panels show representative results of the DNA cleavage activity assays. The asterisks show the 5-³²P-labeled oligonucleotides. (b) Detection of MUTYH proteins in cumate-inducible stable H1299 lung cancer cell lines designed to express MUTYH in the presence of cumate; the MUTYH proteins were detected using a Western blot analysis. Empty vector-transposed cells were used as a control. (c) Proportion of G → T mutation among mutations at position 159 of supF on 8BrG-containing pMY189 plasmids replicated in MUTYH-overexpressing H1299 cells and empty vector-transposed H1299 cells. The total number of mutations at position 159 of supF is shown in parentheses.

(a)

(b)

(c)