Photorepair of RNA polymerase arrest and apoptosis after ultraviolet irradiation in normal and XPB deficient rodent cells

Abstract

Cyclobutane pyrimidine dimers (CPDs) are directly involved in signaling for UV-induced apoptosis in mammalian cells. Failure to remove these lesions, specially those located at actively expressing genes, is critical, as cells defective in transcription coupled repair have increased apoptotic levels. Thus, the blockage of RNA synthesis by lesions is an important candidate event triggering off active cell death. In this work, wild-type and XPB mutated Chinese hamster ovary (CHO) cells expressing a marsupial photolyase, that removes specifically CPDs from the damaged DNA, were generated, in order to investigate the importance of this lesion in both RNA transcription blockage and apoptotic induction. Photorepair strongly recovers RNA synthesis in wild-type CHO cell line, although the resumption of transcription is decreased in XPB deficient cells. This recovery is accompanied by the prevention of cells entering into apoptosis. These results demonstrate that marsupial photolyase has access to CPDs blocking RNA synthesis in vivo, and this may be affected by the presence of a mutated XPB protein.

CPD-photolyase adenovirus-mediated gene transfer in normal and DNA-repair-deficient human cells

“…In light conditions, the specific removal of CPD photoproducts in infected cells was demonstrated by sedimentation in alkaline sucrose gradients and CPD immunostaining, indicating the phr-EGFP fusion protein is functional in photorepair. The heterologous removal of CPDs after PRL exposure clearly increases the clonogenic capacity and prevents induction of apoptosis in UV-irradiated cells, confirming that these photoproducts are important signals to cell death after UV, as previously described (Miyaji and Menck, 1996;Chiganças et al, 2000;Chiganças et al, 2002), and demonstrating that phr-EGFP fusion protein is effective in CPD recognition and photorepair whatever the NER status of the cell. CPDs are a physical hindrance to the transcriptional complex progression (Donahue et al, 1994); the RNA polymerase II complex stalled by persistent photoproducts in the transcribed strand is an active signal to cellular suicide after UV irradiation (Ljungman and Zhang, 1996;Chiganças et al, 2002).…”

“…The heterologous removal of CPDs after PRL exposure clearly increases the clonogenic capacity and prevents induction of apoptosis in UV-irradiated cells, confirming that these photoproducts are important signals to cell death after UV, as previously described (Miyaji and Menck, 1996;Chiganças et al, 2000;Chiganças et al, 2002), and demonstrating that phr-EGFP fusion protein is effective in CPD recognition and photorepair whatever the NER status of the cell. CPDs are a physical hindrance to the transcriptional complex progression (Donahue et al, 1994); the RNA polymerase II complex stalled by persistent photoproducts in the transcribed strand is an active signal to cellular suicide after UV irradiation (Ljungman and Zhang, 1996;Chiganças et al, 2002). The removal of these CPD lesions, by the photolyase fusion enzyme in human cells infected with Adphr-EGFP and exposed to PRL, prevents apoptosis induction probably by eliminating the signal to death in active genes, allowing for the RNA synthesis to restart, and thus promoting progression in the cell cycle and survival.…”

“…The experimental tool of heterologous photorepair in mammalian cells to investigate the role of CPDs in apoptosis has been very informative in different systems (Asashina et al, 1999;Kulms et al, 1999;Chiganças et al, 2000;Stege et al, 2000;Chiganças et al, 2002). CPD-photolyases are enzymes that can recognize and form a stable complex with the CPD in a light-independent manner (Sancar, 1994;Todo, 1999), removing the photoproduct in the presence of visible light, thereby restoring the native state of the bases in the DNA molecule.…”

“…CPD-photolyases are enzymes that can recognize and form a stable complex with the CPD in a light-independent manner (Sancar, 1994;Todo, 1999), removing the photoproduct in the presence of visible light, thereby restoring the native state of the bases in the DNA molecule. By using the heterologous expression of photolyases, we showed that CPDs represent an important signal for UV-induced apoptotic suicide in human cells (Chiganças et al, 2000), mainly those present in the transcribed strand of active genes, by representing a complete block to RNA polymerase II progression (Donahue et al, 1994;Ljungman and Zhang, 1996;Chiganças et al, 2002). In this work, we generated a recombinant adenovirus carrying a CPDphotolyase-EGFP fusion gene, employed to study the behavior of this DNA repair enzyme in human cells.…”

p53 Mutant Human Glioma Cells Are Sensitive to UV-C-Induced Apoptosis Due to Impaired Cyclobutane Pyrimidine Dimer Removal

“…NER-deficient cells are hypersensitive to UV-C-induced apoptosis (13). Also, removal of photolesions by photolyase was shown to prevent apoptosis (14,15), indicating that unrepaired DNA lesions are the main cause of UV-C-induced apoptosis in mammalian cells. The role played by p53 on NER has been studied extensively (for a recent review, see ref.…”