Reduced Repair of Non‐dimer Photoproducts in a Gene Transfected Into Xeroderma Pigmentosum Cells

Abstract

Abstract— 4ells from patients with the sun sensitive cancer‐prone disease, xeroderma pigmentosum (XP) have defective repair of UV damaged DNA with reduced excision of the major photoproduct, the cyclobutane type pyrimidine dimer. Other (non‐dimer) photoproducts, have recently been implicated in UV mutagenesis. Utilizing an expression vector host cell reactivation assay, we studied UV damaged transfecting DNA that was treated by in vitro photoreactivation to reverse pyrimidine dimers while not altering other photoproducts. We found that the reduced expression of a UV damaged transfecting plasmid in XP complementation group A cells is only partially reversed by photoreactivation. E. coli photolyase treatment of pSV2catSVgpt exposed to 100 or 200 J m−2 of 254 nm radiation removed 99% of the T4 endonuclease V sensitive sites. Transfection of XP12BE(SV40) cells with photoreactivated pSV2catSVgpt showed residual inhibition corresponding to 25 to 37% of the lethal hits to the cat gene. This residual inhibition corresponds to the fraction of non‐dimer photoproducts induced by UV. This result implies that XP12BE(SV40) cells do not repair most of the non‐dimer photoproducts in DNA.

Ultraviolet-induced mutations in Cockayne syndrome cells are primarily caused by cyclobutane dimer photoproducts while repair of other photoproducts is normal.

“…The enzyme had no endonucleolytic activity on unirradiated plasmid. UV-irradiated or unirradiated pZ189 was mixed with photolyase and treated as described (25). Briefly, the mixture in glass tubes treated with silane (RepelSilane, LKB) was exposed to 405-nm monochromatic visible light for 1 h. Control plasmid was subjected to the entire PR procedure but the photolyase enzyme was omitted from the reaction buffer.…”

The xeroderma pigmentosum group C gene leads to selective repair of cyclobutane pyrimidine dimers rather than 6-4 photoproducts

“…To generate 6-4PP, plasmid was treated with UVC and subsequently photoreactivated with E. coli photolyase and 405-nm monochromatic light. This procedure is known to remove Ͼ99% of UVC-induced CPD lesions, as shown by agarose gel pattern indicating sensitivity to nicking of supercoiled plasmid form I by treatment with T4 endonuclease V (specifically cleaves at CPD but not 6-4PP sites) (25,30). However, this detection system loses its sensitivity at UVC doses Ͼ30-50 J͞m 2 to plasmid.…”

“…To generate only CPD lesions, the nonreplicating pRSVcat reporter gene plasmid (24,25) was irradiated with 313-nm monochromatic UVB light (2 J͞m 2 per s) in the presence of 10 Ϫ2 M acetophenone (Sigma) for 1 h under anoxic conditions (28,29). To induce only 6-4PP lesions, the plasmid was irradiated with 1,000 J͞m 2 UVC (1.6 J͞m 2 per s) and subsequently treated with photolyase (PharMingen; 1 ml of 0.05 mg͞ml pRSVcat ϩ 1 l of 5 mg͞ml photolyase) and 405-nm monochromatic blue light (4.5 J͞m 2 per s) for 1 h to remove UVC-induced CPD lesions (30,31). Photolesions after treatment were measured with the ELISA technique as described above, except that for CPD detection 1.5 ng of plasmid DNA and for 6-4PP detection 15 ng of plasmid DNA was used.…”

The Association of the Xeroderma Pigmentosum Group D DNA Helicase (XPD) with Transcription Factor IIH Is Regulated by the Cytosolic Iron-Sulfur Cluster Assembly Pathway

“…that fibroblasts derived from xeroderma pigmentosum patients harboring mutations in XPD display increased sensitivity to UV-induced DNA damage due to defects in the NER pathway (21) and this mutant phenotype can be rescued by expression of wild type XPD (22). Utilizing this experimental system, we tested whether the XPD-⌬277-286 mutant that is defective in MMS19 binding is able to complement the UV sensitivity phenotype of XPD-deficient fibroblasts.…”