8-Oxoguanine-mediated transcriptional mutagenesis causes Ras activation in mammalian cells

Abstract

8-Oxoguanine (8OG) is efficiently bypassed by RNA polymerases in vitro and in bacterial cells in vivo, leading to mutant transcripts by directing incorporation of an incorrect nucleotide during transcription. Such transcriptional mutagenesis (TM) may produce a pool of mutant proteins. In contrast, transcription-coupled repair safeguards against DNA damage, contingent upon the ability of lesions to arrest elongating RNA polymerase. In mammalian cells, the Cockayne syndrome B protein (Csb) mediates transcription-coupled repair, and its involvement in the repair of 8OG is controversial. The DNA glycosylase Ogg1 initiates base excision repair of 8OG, but its influence on TM is unknown. We have developed a mammalian system for TM in congenic mouse embryonic fibroblasts (MEFs), either WT or deficient in Ogg1 (ogg(-/-)), Csb (csb(-/-)), or both. This system uses expression of the Ras oncogene in which an 8OG replaces guanine in codon 61. Repair of 8OG restores the WT sequence; however, bypass and misinsertion opposite this lesion during transcription leads to a constitutively active mutant Ras protein and activation of downstream signaling events, including increased phosphorylation of ERK kinase. Upon transfection of MEFs with replication-incompetent 8OG constructs, we observed a marked increase in phospho-ERK in ogg(-/-) and csb(-/-)ogg(-/-) cells at 6 h, indicating persistence of the lesion and the occurrence of TM. This effect is absent in WT and csb(-/-) cells, suggesting rapid repair. These studies provide evidence that 8OG causes TM in mammalian cells, leading to a phenotypic change with important implications for the role of TM in tumorigenesis.

Fig. 1.

Mammalian TM system. WT, codon 61 transcription results in a glutamine (Q) upon translation (Left). However, if an 8OG residue (*) replaces G at the first base position, it can direct the incorporation of either C or A into the nascent mRNA (Right). If a C is incorporated, the resulting translation product will be a WT Ras with Q at position 61. However, if an A is incorporated, the Ras generated will contain a lysine (K) at this position and be constitutively active, resulting in an elevation in cellular levels of phosphorylated ERK (P-ERK). Codons and corresponding amino acids flanking position 61 are also indicated. NTS, nontranscribed strand; TS, transcribed strand.

Fig. 2.

8OG causes TM in ogg^−/− and csb^−/−ogg^−/− cells. (A) Representative Western analysis of lysates from transfected cells. Transfections were done in duplicate. Specific antibodies were used to detect P-ERK, Ras, and total ERK (Materials and Methods). (B) Bands from Western blots were quantified and normalized as described in the text. P-ERK levels relative to those obtained with the mutant construct (set to 100% for each cell line) are plotted. Error bars represent standard deviations from six to eight independent transfection experiments. *Signal obtained with 8OG construct was statistically significantly different from that observed with the normal construct (P < 0.05), as assessed by Student's t test. Differences between the mutant construct and normal construct were significant in all cell lines.

Fig. 3.

ogg^−/− and csb^−/−ogg^−/− cells are impaired in their repair of 8OG. (A) Representative Southern blot of reisolated construct digested with Fpg to assess the residual levels of unrepaired 8OG at various times after transfection. (B) Quantification of the data (two independent experiments). Error bars represent standard deviations.

Fig. 4.

Proposed role for TM in tumorigenesis. DNA damage (jagged symbol), either spontaneous or induced, occurs within the coding region of an oncogene (e.g., Ras) on the transcribed strand. Transcriptional bypass of this lesion leads to a population of mutant mRNA that, upon translation, gives rise to mutant protein. The resulting mutant protein is capable of signaling in the absence of appropriate stimuli, activating the MAPK cascade and downstream genes that mediate cell proliferation. This signaling promotes a round of DNA replication over the same, unrepaired DNA lesion, allowing for the fixation of a permanently heritable mutation (red dot) at the site of the damage. Upon cell division, one daughter cell will inherit this mutation and will be able to pass it on to progeny regardless of whether the damage is subsequently repaired, giving rise to an aberrant, dividing cell population that, with appropriate additional genetic changes, ultimately develops into a tumor.