Action-at-a-distance mutations induced by 8-oxo-7,8-dihydroguanine are dependent on APOBEC3.

DNA oxidation is a serious threat to genome integrity and is involved in mutations and cancer initiation. The G base is most frequently damaged, and 8-oxo-7,8-dihydroguanine (GO, 8-hydroxyguanine) is one of the predominant damaged bases. In human cells, GO causes a G:C→T:A transversion mutation at the modified site, and also induces untargeted substitution mutations at the G bases of 5'-GpA-3' dinucleotides (action-at-a-distance mutations). The 5'-GpA-3' sequences are complementary to the 5'-TpC-3' sequences, the preferred substrates for apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3 (APOBEC3) cytosine deaminases, and thus their contribution to mutagenesis has been considered. In this study, APOBEC3B, the most abundant APOBEC3 protein in human U2OS cells, was knocked down in human U2OS cells, and a GO-shuttle plasmid was then transfected into the cells. The action-at-a-distance mutations were reduced to ~25% by the knockdown, indicating that GO-induced action-at-a-distance mutations are highly dependent on APOBEC3B in this cell line.

Biased distribution of action-at-a-distance mutations by 8-oxo-7,8-dihydroguanine.

8-Oxo-7,8-dihydroguanine (8-hydroxyguanine, G°) is a major oxidized base that is considered to play pivotal roles in the pathogenesis of various diseases, including cancer. G° induces G:C → T:A transversions at the damage site and untargeted (action-at-a-distance) mutations of G bases at 5'-GpA sequences. In this study, we examined the distribution of the action-at-a-distance mutations and the effects of the replication origin position relative to G° on the untargeted mutagenesis. The G° base was introduced into two shuttle plasmids, each with the SV40 replication origin at a different position with respect to the supF gene. The oxidized base was located at an upstream or downstream site (outside of the gene), or the center of the region encoding the pre-tRNA sequence of the gene, in the sense strand. These shuttle plasmids were introduced into human U2OS cells. The action-at-a-distance mutations were more frequently induced when the G° base was located downstream of the supF gene than upstream of the gene. In addition, more action-at-a-distance mutations were observed when the SV40 origin was present on the 5'-side of the G° base. These results indicated that the action-at-a-distance mutations are predominantly induced on the 5'-side of the lesion and occurred more frequently when the damaged base was located on the lagging strand template.

Paradoxical role of the major DNA repair protein, OGG1, in action-at-a-distance mutation induction by 8-oxo-7,8-dihydroguanine.

Oxidatively damaged bases induce mutations and are involved in cancer initiation. 8-Oxo-7,8-dihydroguanine (G°, 8-hydroxyguanine) is an abundant oxidized base that induces targeted G:C→T:A transversions in human cells, as well as untargeted base substitution (action-at-a-distance) mutations of the G bases of 5'-GpA-3' dinucleotides. The action-at-a-distance mutations become more frequent than the targeted transversions when the amount of Werner syndrome (WRN) protein is decreased. In this study, OGG1, the major DNA glycosylase for the damaged base, and WRN were knocked down in isolation and in combination in human U2OS cells, and a shuttle plasmid carrying G° was introduced into the knockdown cells. Interestingly, fewer action-at-a-distance mutations were observed in the WRN plus OGG1 double knockdown cells, as compared to the WRN single knockdown cells. These results indicated the paradoxical role of OGG1, as an accelerator of the action-at-a-distance mutations by the oxidized guanine base.

New indicator Escherichia coli strain for rapid and accurate detection of supF mutations.

BACKGROUND: The supF gene of Escherichia coli is useful for forward mutation analysis in bacterial and mammalian cells used in mutagenesis and DNA repair studies. Indicator E. coli strains, such as KS40/pOF105, have been used to analyze supF mutations. However, KS40/pOF105 is not enough to select supF mutants on nutrient-rich agar plates. Therefore, in this study, a new indicator E. coli strain for rapid and accurate detection of supF mutations was developed. RESULTS: The gyrA and rpsL genes with an amber mutation were integrated into the chromosomal DNA of E. coli KS40 to produce a new indicator strain, RF01. RF01 cells transformed by the wild-type supF gene were sensitive to nalidixic acid and streptomycin on LB agar plates. supF mutant frequencies and mutation spectra in RF01 were similar to those in KS40/pOF105. In addition, some mutations in supF were only detected in RF01. CONCLUSION: RF01 is a new and useful indicator E. coli strain for analyzing supF mutations.