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  • Our supF forward mutation assay revealed for the first time

    2021-11-25

    Our supF forward mutation assay revealed, for the first time, that 5OHU predominantly induced the C→T mutation in human cells. The mechanism underlying the induction of the C→T mutation in human cells by 5OHU remains unclear, however, the finding that 5OHU predominantly induced the C→T mutation was also seen in E. coli [24]. Since the C→T (G:C to A:T) mutation is the most frequent or second most frequent mutation type in many types of human cancers [44], it is considered that 5OHU could be involved in the mutation spectrum observed in human cancers. In addition, C→T (G:C to A:T) mutation is reported as the most predominant among mutations detected in the carcinomas in patients with NAP, which is in contrast to the predominance of the G:C to T:A mutation in the carcinomas in MAP patients [2], [4]. Since our study has revealed that the C→T mutation is induced by 5OHU in human cells and that NTHL1 has the ability to suppress the increase in the mutation frequency induced by 5OHU, the C→T mutation seen in NAP-associated carcinomas could be caused, at least in part, by 5OHU. This has an important implication, since no definite modified bases causing a C→T mutation in NAP-associated carcinomas have been found so far. However, there is one caution: it is possible that Phenacetin modifications other than 5OHU could also cause a Phenacetin C→T mutation in such carcinomas, and the actual extent of the involvement of 5OHU in inducing this mutation remains to be precisely clarified. With regard to the NTHL1 variants R19Q, V179I, V217F, and G286S, this study showed their retained ability for the repair of three kinds of DNA substrates, i.e., 5OHU:G, DHU:G, and Tg:A mismatch-containing DNA. These results surprised us at first, because prediction using the PolyPhen-2, SIFT, and PROVEAN software suggested that the four missense NTHL1 variants were functionally damaged in most cases; this implies discrepancies in the results between the DNA cleavage activity assay and results of the prediction. In this context, Miosge et al. [45] recently compared the predicted and actual consequences of missense mutations and reported that only 20% of mutations predicted to be deleterious by the PolyPhen-2 software showed a discernible phenotype in individual homozygotes. Therefore, we consider that the prediction for the R19Q, V179I, V217F, and G286S NTHL1 variants using above-mentioned software was not adequate in most cases and that the NTHL1 alleles encoding these variants were not associated with the development of NAP. We would like to emphasize here that practical functional evaluation is important to make a precise judgment on the pathogenicity of NTHL1 variants for NAP. The DNA cleavage activity assay in the present study clearly demonstrated the 5OHU DNA glycosylase activities of SMUG1, NEIL1, TDG, UNG2, and NTHL1, and the supF forward mutation assay showed, for the first time, that the activity of each of the proteins was compatible with its ability to suppress 5OHU-induced mutations. Our findings were in good agreement with the 5OHU DNA glycosylase activities of SMUG1, NEIL1, UNG2, and NTHL1 in previous studies [12], [25], [26], [27], [28]. With regard to TDG, Hardeland et al. [46] described that 5OHU mispaired with G was excised from DNA by TDG, although they did not provided no experimental data. Thus, experimental data on the 5OHU DNA glycosylase activity of TDG was shown for the first time in our study, and our findings on the 5OHU DNA glycosylase activity of TDG were similar to those reported by Hardeland et al. [46]. In total, five kinds of DNA glycosylases were shown to be involved in the repair of 5OHU. A search of the literature for reports on the repair of other damaged bases by DNA glycosylases revealed that it is not uncommon for a damaged base to be repaired by multiple kinds of DNA glycosylases; e.g., 3,N-ethenocytosine and Tg [11], [47], [48], [49]. The fact implies that avoidance of damaged base-induced mutations or strand synthesis block by the base excision repair system is important in human cells.