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

Abstract

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

“…1D, we can observe that the CPDphotolyase was effective in CPD photorepair in PRL conditions, generating locally UV-irradiated spots containing only 6-4PPs. After PRL exposure, the homogeneous distribution of the CPDphotolyase throughout the nucleus is restored between 1 and 4 hours after local UV (25). In addition, we cannot exclude that the permanence of the CPD photolyase at these sites can be due to the presence of 6-4PP lesions after CPD photorepair because some cross-reactivity between CPD-photolyase and 6-4PP has been reported (35).…”

“…The cells were grown as described (25). The human primary cells used in this work, 405VI (normal), TTD1VI (XPD mutated), TTD9VI (XPD mutated), the XPD-complemented TTD1VI and TTD9VI, XP22VI (XPD mutated), and SV40-TTD1VI, were from our laboratory (26,27).…”

“…Primary fibroblasts in coverslips were infected with two different purified adenoviruses: one carrying a CPD-photolyase from marsupial (rat kangaroo, Potorous tridactylus) as a fusion protein with the reporter protein EGFP (CPD-photolyase-EGFP, vector AdCPDphr; ref. 25), and a second one carrying a 6-4PP-photolyase from plant (Arabidopsis thaliana) coexpressed with EGFP (6-4PP-photolyase-IRES-EGFP, vector Ad6-4phr; ref. 29).…”

“…Different cell lines plated on coverslips and treated with different adenovirus were submitted to local UV irradiation (UV-C, 254 nm) as described (30). Photoreactivation was done as described (25). For treatment with TSA (Sigma Aldrich), the inhibitor was added to the medium at a concentration of 10 ng/mL during the photoreactivating light (PRL) incubation.…”

Recombinant Viral Vectors for Investigating DNA Damage Responses and Gene Therapy of Xeroderma Pigmentosum

“…These successful studies have motivated the adenoviruses-mediated expression of the CPD-photolyase from the rat kangaroo Potorous tridactylus and the plant 6-4PP-photolyase from Arabidopsis thaliana in human cells aiming to discriminate the precise role of UV-induced cellular responses in both NER-deficient and NER-proficient human cells. Employing immunofluorescence, immunoblot and local UV experiments, it has been possible to see that these enzymes are not only very specific for their lesions, but are also really fast to find them, colocalizing with regions of damaged DNA and other DNA repair enzymes in less than two minutes (Chiganças et al, 2004;Lima-Bessa et al, 2008). Adenoviral-mediated photorepair of CPDs substantially prevented apoptosis in all UVirradiated cell lines (both NER-deficient and NER-proficient cells), confirming the involvement of these lesions in cell death signaling, as previously reported.…”