Vanessa Chiganças

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

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

Cyclobutane pyrimidine dimers (CPDs) are the most frequent and deleterious lesions generated in the mammalian genome after UV-C irradiation. The persistence of these lesions in DNA can be toxic and mutagenic, and also represents a specific signal to apoptosis. To investigate the CPDs repair in situ and consequent UV-induced apoptosis in human cells, we generated a recombinant adenovirus vector containing the gene encoding a CPD-photolyase-EGFP fusion protein (Adphr-EGFP). Adphr-EGFP-infected cells are proficient in photorepair, which prevents apoptotic cell death in comparison with samples kept in the dark, indicating that the fusion protein is functional in CPD recognition and removal. By using local UV irradiation, foci of the photolyase fusion protein were observed in UV-damaged areas of the nuclei in colocalization with NER enzymes. Phr-EGFP migration to CPD sites and redistribution after photorepair was followed, and shown to present similar kinetics in normal or DNA-repair-deficient cells. To our knowledge, this is the first report of an investigation of CPDs repair in situ employing a CPD-photolyase-EGFP enzyme. The Adphr-EGFP vector can be an informative tool to investigate the repair and cellular consequences of UV-induced lesions in primary human cells.

Defective transcription/repair factor IIH recruitment to specific UV lesions in trichothiodystrophy syndrome.

Most trichothiodystrophy (TTD) patients present mutations in the xeroderma pigmentosum D (XPD) gene, coding for a subunit of the transcription/repair factor IIH (TFIIH) complex involved in nucleotide excision repair (NER) and transcription. After UV irradiation, most TTD/XPD patients are more severely affected in the NER of cyclobutane pyrimidine dimers (CPD) than of 6-4-photoproducts (6-4PP). The reasons for this differential DNA repair defect are unknown. Here we report the first study of NER in response to CPDs or 6-4PPs separately analyzed in primary fibroblasts. This was done by using heterologous photorepair; recombinant adenovirus vectors carrying photolyases enzymes that repair CPD or 6-4PP specifically by using the energy of light were introduced in different cell lines. The data presented here reveal that some TTD/XPD mutations affect the recruitment of TFIIH specifically to CPDs, but not to 6-4PPs. This deficiency is further confirmed by the inability of TTD/XPD cells to recruit, specifically for CPDs, NER factors that arrive in a TFIIH-dependent manner later in the NER pathway. For 6-4PPs, we show that TFIIH complexes carrying an NH(2)-terminal XPD mutated protein are also deficient in recruitment of NER proteins downstream of TFIIH. Treatment with the histone deacetylase inhibitor trichostatin A allows the recovery of TFIIH recruitment to CPDs in the studied TTD cells and, for COOH-terminal XPD mutations, increases the repair synthesis and survival after UV, suggesting that this defect can be partially related with accessibility of DNA damage in closed chromatin regions.

Involvement of DNA replication in ultraviolet-induced apoptosis of mammalian cells

Exposure of cells to ultraviolet (UV) light damages the genome and the persistence of DNA lesions triggers apoptosis in mammalian cells. RNA transcription blockage by DNA damage is believed to be implicated in signaling for UV-induced apoptosis, but the role played by DNA replication in this process is still unclear. To address this point, we have employed the DNA polymerase inhibitor aphidicolin in UV-irradiated wild-type and XPB-mutated Chinese hamster ovary cells. The data obtained with synchronized cells indicate that induction of apoptosis by UV light is independent of the cell cycle phase. Nevertheless, cells treated with aphidicolin after UV exposure showed a significant prevention of apoptosis induction when compared to proliferating cells. These results were observed in both DNA-repair proficient and deficient cells, indicating that the prevention of apoptosis by aphidicolin is independent of the cells’ ability to repair the photolesions caused by UV. Taken together, these data suggest that replication of damaged DNA also leads to critical events signaling for UV-induced cell death.

Exploring DNA damage responses in human cells with recombinant adenoviral vectors

Recombinant adenoviral vectors provide efficient means for gene transduction in mammalian cells in vitro and in vivo. We are currently using these vectors to transduce DNA repair genes into repair deficient cells, derived from xeroderma pigmentosum (XP) patients. XP is an autosomal syndrome characterized by a high frequency of skin tumors, especially in areas exposed to sunlight, and, occasionally, developmental and neurological abnormalities. XP cells are deficient in nucleotide excision repair (affecting one of the seven known XP genes, xpa to xpg) or in DNA replication of DNA lesions (affecting DNA polymerase eta, xpv). The adenovirus approach allows the investigation of different consequences of DNA lesions in cell genomes. Adenoviral vectors carrying several xp and photolyases genes have been constructed and successfully tested in cell culture systems and in vivo directly in the skin of knockout model mice. This review summarizes these recent data and proposes the use of recombinant adenoviruses as tools to investigate the mechanisms that provide protection against DNA damage in human cells, as well as to better understand the higher predisposition of XP patients to cancer. Human & Experimental Toxicology (2007) 26, 899—906