Transcriptional mutagenesis mediated by 8-oxoG induces translational errors in mammalian cells

Reactive oxygen species formed within the mammalian cell can produce 8-oxo-7,8-dihydroguanine (8-oxoG) in mRNA, which can cause base mispairing during gene expression. Here we found that administration of 8-oxoGTP in MTH1-knockdown cells results in increased 8-oxoG content in mRNA. Under this condition, an amber mutation of the reporter luciferase is suppressed. Using second-generation sequencing techniques, we found that U-to-G changes at preassigned sites of the luciferase transcript increased when 8-oxoGTP was supplied. In addition, an increased level of 8-oxoG content in RNA induced the accumulation of aggregable amyloid β peptides in cells expressing amyloid precursor protein. Our findings indicate that 8-oxoG accumulation in mRNA can alter protein synthesis in mammalian cells. Further work is required to assess the significance of these findings under normal physiological conditions.

Structural analysis of base mispairing in DNA containing oxidative guanine lesion

Classical molecular dynamics methods were used to analyze the importance of 8-oxoguanine (8-oxoG) pairing with other DNA bases in order to determine the impact of oxidative guanine lesions on DNA structure. Six lesioned molecules, each containing 8-oxoG mispaired with one of the four normal bases on the the opposite strand at the center of 40-mer DNA, and one non-damaged DNA molecule, were simulated for 2 nanoseconds of real time. The 8-oxoG lesioned bases were found to incorporate opposite all normal bases. There are observed conformational and energetical differences among these parings. 8-oxoG in anti-form creates firm hydrogen bonds with cytosine and this bonding has a strong attractive electrostatic interaction energy similar to that of a native base pair-guanine to cytosine. Meanwhile, it does not form a stable base pair with purine bases (adenine and guanine) nor with the pyrimidine base thymine. On the other hand, the 8-oxoG in syn-form was found to pair with adenine.

Sequence-Directed Base Mispairing in Human Oncogenes

ABSTRACT The most frequently observed mutations in ras oncogenes in solid human tumors are GC→AT transitions at the 3′ G residue of the GG doublet in codon 12 of these oncogenes. We had shown previously that mutagenesis by thymidine occurred with the same sequence specificity in mammalian cells, in that mutagenesis occurred preferentially at the 3′ G of GG doublets. In this study, in vitro DNA synthesis experiments were carried out to assess the effect of local DNA sequence on base mispairing in order to determine the mechanism of sequence-directed mutagenesis by thymidine and its possible relationship to activating point mutations in N-, Ki- and Ha-ras oncogenes in solid human tumors. To avoid complicating the interpretation of the results because of the occurrence of mismatch repair as well as base misincorporation, the experiments were carried out in a repair-free environment with exonuclease-free Klenow polymerase. The results of these experiments showed that misincorporation of deoxyribosylthymine (dT) occurred with several-fold-greater efficiency opposite the 3′ G compared to the 5′ G of the GG doublet in codon 12 of human ras oncogenes. These results further demonstrated that the relative difference in the extent of dT misincorporation opposite the 3′ G and the 5′ G of GG doublets in codon 12 in the various ras oncogenes was affected by the base immediately upstream of the doublet. Within the GG doublet, it was seen that the 5′ G and 3′ G residues had an effect on the extent of dT misincorporation opposite each other. The 5′ G was shown to have a stimulatory effect on dT misincorporation opposite the 3′ G, while the 3′ G was shown to have an inhibitory effect on dT misincorporation opposite the 5′ G. Presumably, these mutual interactions within GG doublets are additive, such that the large differential in dT misincorporation observed between the 3′ G and 5′ G residues in GG doublets is the end result of the combined stimulatory and inhibitory effects within these doublets. Since the observed pattern of dT misincorporation within GG doublets corresponds to the most frequent mode of activation of ras oncogenes in solid human tumors, the results of these experiments suggest that sequence-directed dT misincorporation may be involved in the pattern of activation of humanras oncogenes, by causing GC→AT transitions preferentially at the 3′ G of the GG doublet in codon 12 of these oncogenes.