Figure 1.
Ppluc gene modification inducing transcriptional mutagenesis and phenotypical change in mammalian cells.
Five codons of the Ppluc gene were modified, to investigate the effects of the presence of an 8OG residue in the transcribed strand (TS) of a gene. (A) Codons 5, 297 and 445 were modified to specify lysine (K) (LFS/Lys), a stop codon (LFS/Stop) or lysine through transcriptional mutagenesis (TM) and glutamine (Gln) through faithful transcription (8OG/Stop). (B) Codon 344 was modified to encode glutamic acid (LFS/Glu)344 or alanine (LFS/Ala)344 leading to the production of an active or an inactive E344A Ppluc, respectively. Codon 422 was modified to specify either aspartic acid (LFS/Asp)422 or alanine (LFS/Ala)422, leading to the production of an active or an inactive D422A Ppluc, respectively. In (8OG/Ala) constructs, transcriptional bypass of the 8OG leads to the production of active Ppluc through TM or the production of inactive Ppluc through faithful transcription.
Table 1.
Cell lines used in this study.
Table 2.
Relative luciferase activity 24 h after transfection of the various cell lines with constructs with lesions at codons 5, 297 and 445.
Table 3.
Relative luciferase activity 24 h after the transfection of various cell lines with constructs with lesions at codons 344 and 422.
Figure 2.
8OG-driven transcriptional mutagenesis in human cells.
We provide here a schematic diagram of the fate of an 8OG:T mispair at codon 445. After transfection with (8OG/Stop)445, the DNA molecule can be repaired by various DNA repair pathways (see text for details). Depending on the pathway, transcription of the repaired molecules leads to the production of an mRNA molecule containing either a C or a U as the first base of codon 445, thus generating a stop codon or encoding a glutamine, respectively. The transcription of unrepaired molecules is the source of 8OG-driven TM events. We assessed the frequency of such events by extracting total RNA from MRC5V1 cells transfected with the (8OG/Stop)445 construct 24 hours after transfection. A portion of the Ppluc mRNA was amplified and RT-PCR fragments were subcloned into pUC18 for sequencing. The numbers of each type of cDNA are indicated for each type of base insertion.
Figure 3.
The pattern of 8OG-driven phenotypical change over time.
MRC5 cells were transfected with (LFS/Glu)344 (open triangles, solid lines), (LFS/Asp)422 (open circles, solid lines), (8OG/Ala)344 (closed triangles, solid lines), (8OG/Ala)422 (closed circles, solid lines), (LFS/Ala)344 (open triangles, dashed lines) or (LFS/Ala)422 (open circles, dashed lines). Ppluc and Rrluc activities in transfected cells were quantified at different time points after transfection at time 0. Each experimental point corresponds to the mean of six replicates±the standard error of the mean. RLU: relative light units.
Figure 4.
Speculative biological outcomes of TM.
In normal conditions (left part), the expression of a gene in non-dividing cells results in the production of normal proteins. When a DNA lesion (e.g. 8OG) occurs in the transcribed strand of a gene (on the right), multiple RNA polymerase bypasses of the lesion result in misincorporation events (e.g. A instead of C opposite to 8OG, open circle on the mRNA) at the same position in most of the mRNA molecules produced before the DNA is repaired. A large population of mutated mRNA molecules can then be translated multiple times to generate large amounts of mutated protein, which may induce a transient phenotypical change. However, if the mutated proteins are resistant to protein degradation and have a dominant effect, the phenotypical change may be prolonged or even permanent. For example, the mutated proteins may be more likely to form aggregates, providing a nucleation point for the recruitment of normal proteins produced after the DNA lesion is repaired. These protein aggregates may therefore mimic a dominant-negative effect ultimately resulting in cell degeneration, as observed in neurodegenerative diseases.