To elucidate the mutagenicity of 8BrG in human cells, a supF forward mutation assay with a shuttle plasmid, pMY189, was performed in two human tumor cell lines (H1299 and LN428) and a human normal cell line (16HBE14o-).
A similar result was obtained when a pMY189 plasmid containing an 8BrG residue at position 144 of the supF gene was used for a supF forward mutation assay in H1299 cells (Supplementary Figure S1).
We established human H1299 lung cancer cells capable of inducibly expressing MUTYH using the PiggyBac transposon vector system (Figure 4(b)) and performed a supF forward mutation assay using a pMY189 plasmid containing an 8BrG at position 159 of the supF gene in the established cell lines.
In this study, we performed a supF forward mutation assay using a shuttle vector plasmid containing a single 8BrG residue in three kinds of human cells and revealed that 8BrG in DNA predominantly induces a G [right arrow] T mutation but can also induce G [right arrow] C, G [right arrow] A, and delG mutations in human cells.
According to previous supF forward mutation assays using wild-type pMY189 in human cells [31, 32], a G:C to T:A mutation was the most predominant mutation type among base substitution mutations at any G positions in the supF gene on untreated pMY189, although G:C to C:G and G:C to A:T mutations were also detected at lower frequencies (Supplementary Table S3).
These assays were based on a forward mutation fidelity assay developed by Kunkel and colleagues, which used a gap-filling reaction with a DNA polymerase on a lacZ template sequence, followed by ligation and transformation into E.
That study used a forward mutation assay (not PCR), expressed fidelity simply as the ratio of white colonies to blue with no accounting for PCR amplification efficiency, and used experimental conditions ([Mg.sup.2+] concentration) that differ significantly from typical PCR conditions.
To investigate the ability of MUTYH p.Arg81Trp variant to suppress mutations caused by 8OHG in human cells, we planned to use the piggyBac transposon vector system  to establish human cells capable of inducibly expressing MUTYH protein and to perform a supF forward mutation assay using the shuttle plasmid pMY189, which contains a single 8OHG in the supF gene.
Next, the mutation frequencies were compared among the empty vector-transposed human cells and the cumate-inducible stable cells expressing WT or a variant MUTYH using a supF forward mutation assay with the shuttle plasmid pMY189.
We further investigated what kind of mutation is contained in the supF mutant colony in the supF forward mutation assay.
Therefore, we investigated type 2 p.Arg81Trp MUTYH using a DNA cleavage assay and a supF forward mutation assay and found that the abilities of p.Arg81Trp to cleave A:8OHG-containing DNA and to suppress mutations caused by 8OHG were severely reduced.
As a result, an impaired cleavage activity of type 2 p.Arg81Trp towards A:8OHG-containing DNA was clearly demonstrated using a DNA cleavage assay, and a severely reduced activity of the protein to suppress mutations caused by 8OHG in human cells was also clearly revealed using a supF forward mutation assay.