CPDs and 6-4PPs play different roles in UV-induced cell death in normal and NER-deficient human cells.

Ultraviolet (UV) light generates two major DNA lesions: cyclobutane pyrimidine dimers (CPDs) and pyrimidine-(6-4)-pyrimidone photoproducts (6-4PPs), but the specific participation of these two lesions in the deleterious effects of UV is a longstanding question. In order to discriminate the precise role of unrepaired CPDs and 6-4PPs in UV-induced responses triggering cell death, human fibroblasts were transduced by recombinant adenoviruses carrying the CPD-photolyase or 6-4PP-photolyase cDNAs. Both photolyases were able to prevent UV-induced apoptosis in cells deficient for nucleotide excision repair (NER) to a similar extent, while in NER-proficient cells UV-induced apoptosis was prevented only by CPD-photolyase, with no effects observed when 6-4PPs were removed by the specific photolyase. These results strongly suggest that both CPDs and 6-4PPs contribute to UV-induced apoptosis in NER-deficient cells, while in NER-proficient cells, CPDs are the only lesions responsible for UV-killing, probably due to the rapid repair of 6-4PPs by NER. As a consequence, the difference in skin photosensitivity, including carcinogenesis, of most of the xeroderma pigmentosum patients and of normal people is probably not only a quantitative aspect, but depends on the type of DNA damage induced by sunlight and its rate of repair.

Adenovirus mediated transduction of the human DNA polymerase eta cDNA.

Xeroderma pigmentosum (XP) is an autosomal recessive photosensitive disorder with an extremely high incidence of skin cancers. Seven complementation groups, corresponding to seven proteins involved in nucleotide excision repair (NER), are associated with this syndrome. However, in XP variant patients, the disorder is caused by defects in DNA polymerase eta; this error prone polymerase, encoded by POLH, is involved in translesion DNA synthesis (TLS) on DNA templates damaged by ultraviolet light (UV). We constructed a recombinant adenovirus carrying the human POLH cDNA linked to the EGFP reporter gene (AdXPV-EGFP) and infected skin fibroblasts from both XPV and XPA patients. Twenty-four hours after infection, the DNA polymerase eta-EGFP fusion protein was detected by Western blot analysis, demonstrating successful transduction by the adenoviral vector. Protein expression was accompanied by reduction in the high sensitivity of XPV cells to UV, as determined by cell survival and apoptosis-induction assays. Moreover, the pronounced UV-induced inhibition of DNA synthesis in XPV cells and their arrest in S phase were attenuated in AdXPV-EGFP infected cells, confirming that the transduced polymerase was functional. However, over-expression of polymerase eta mediated by AdXPV-EGFP infection did not result in enhancement of cell survival, prevention of apoptosis, or higher rate of nascent DNA strand growth in irradiated XPA cells. These results suggest that TLS by DNA polymerase eta is not a limiting factor for recovery from cellular responses induced by UV in excision-repair deficient fibroblasts.

Restoring DNA repair capacity of cells from three distinct diseases by XPD gene-recombinant adenovirus.

The nucleotide excision repair (NER) is one of the major human DNA repair pathways. Defects in one of the proteins that act in this system result in three distinct autosomal recessive syndromes: xeroderma pigmentosum (XP), Cockayne syndrome (CS) and trichothiodystrophy (TTD). TFIIH is a nine-protein complex essential for NER activity, initiation of RNA polymerase II transcription and with a possible role in cell cycle regulation. XPD is part of the TFIIH complex and has a helicase function, unwinding the DNA in the 5' --> 3' direction. Mutations in the XPD gene are found in XP, TTD and XP/CS patients, the latter exhibiting both XP and CS symptoms. Correction of DNA repair defects of these cells by transducing the complementing wild-type gene is one potential strategy for helping these patients. Over the last years, adenovirus vectors have been largely used in gene delivering because of their efficient transduction, high titer, and stability. In this work, we present the construction of a recombinant adenovirus carrying the XPD gene, which is coexpressed with the EGFP reporter gene by an IRES sequence, making it easier to follow cell infection. Infection by this recombinant adenovirus grants full correction of SV40-transformed and primary skin fibroblasts obtained from XP-D, TTD and XP/CS patients.