Efficient repair of cyclobutane pyrimidine dimers at mutational hot spots is restored in complemented Xeroderma pigmentosum group C and trichothiodystrophy/xeroderma pigmentosum group D cells

Xeroderma pigmentosum (XP) and trichothiodystrophy (TTD) are rare heritable diseases. Patients suffering from XP and 50% of TTD afflicted individuals are photosensitive and have a high susceptibility to develop skin tumors. One solution to alleviating symptoms of these diseases is to express the deficient cDNAs in patient cells as a form of gene therapy. XPC and TTD/XPD cell lines were complemented using retroviral transfer. Expressed wild-type XPC or XPD cDNAs in these cells restored the survival to UVC radiation to wild-type levels in the respective complementation groups. Although complemented XP cell lines have been studied for years, data on cyclobutane pyrimidine dimer (CPD) repair in these cells at different levels are sparse. We demonstrate that CPD repair is faster in the complemented lines at the global, gene, strand specific, and nucleotide specific levels than in the original lines. In both XPC and TTD/XPD complemented lines, CPD repair on the non-transcribed strand is faster than that for the MRC5SV line. However, global repair in the complemented cell lines and MRC5SV is still slower than in normal human fibroblasts. Despite the slower global repair rate, in the complemented XPC and TTD/XPD cells, almost all of the CPDs at "hotspots" for mutation in the P53 tumor database are repaired as rapidly as in normal human fibroblasts. Such evaluation of repair at nucleotide resolution in complemented nucleotide excision repair deficient cells presents a crucial way to determine the efficient re-establishment of function needed for successful gene therapy, even when full repair capacity is not restored.     

In vivo recruitment of XPC to UV-induced cyclobutane pyrimidine dimers by the DDB2 gene product

The initial step in mammalian nucleotide excision repair (NER) of the major UV-induced photoproducts, cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4PPs), requires lesion recognition. It is believed that the heterodimeric proteins XPC/hHR23B and UV-DDB (UV-damaged DNA binding factor, composed of the p48 and p127 subunits) perform this function in genomic DNA, but their requirement and lesion specificity in vivo remains unknown. Using repair-deficient xeroderma pigmentosum (XP)-A cells that stably express photoproduct-specific photolyases, we determined the binding characteristics of p48 and XPC to either CPDs or 6-4PPs in vivo. p48 localized to UV-irradiated sites that contained either CPDs or 6-4PPs. However, XPC localized only to UV-irradiated sites that contained 6-4PPs, suggesting that XPC does not efficiently recognize CPDs in vivo. XPC did localize to CPDs when p48 was overexpressed in the same cell, signifying that p48 activates the recruitment of XPC to CPDs and may be the initial recognition factor in the NER pathway.     

DDB accumulates at DNA damage sites immediately after UV irradiation and directly stimulates nucleotide excision repair.

Damaged DNA-binding protein, DDB, is a heterodimer of p127 and p48 with a high specificity for binding to several types of DNA damage. Mutations in the p48 gene that cause the loss of DDB activity were found in a subset of xeroderma pigmentosum complementation group E (XP-E) patients and have linked to the deficiency in global genomic repair of cyclobutane pyrimidine dimers (CPDs) in these cells. Here we show that with a highly defined system of purified repair factors, DDB can greatly stimulate the excision reaction reconstituted with XPA, RPA, XPC.HR23B, TFIIH, XPF.ERCC1 and XPG, up to 17-fold for CPDs and approximately 2-fold for (6-4) photoproducts (6-4PPs), indicating that no additional factor is required for the stimulation by DDB. Transfection of the p48 cDNA into an SV40-transformed human cell line, WI38VA13, was found to enhance DDB activity and the in vivo removal of CPDs and 6-4PPs. Furthermore, the combined technique of recently developed micropore UV irradiation and immunostaining revealed that p48 (probably in the form of DDB heterodimer) accumulates at locally damaged DNA sites immediately after UV irradiation, and this accumulation is also observed in XP-A and XP-C cells expressing exogenous p48. These results suggest that DDB can rapidly translocate to the damaged DNA sites independent of functional XPA and XPC proteins and directly enhance the excision reaction by core repair factors.     

Cyclobutane pyrimidine dimers are responsible for the vast majority of mutations induced by UVB irradiation in mammalian cells

The most prevalent DNA lesions induced by UVB are the cyclobutane pyrimidine dimers (CPDs) and the pyrimidine (6-4) pyrimidone photoproducts ((6-4)PPs). It has been a long standing controversy as to which of these photoproduct is responsible for mutations in mammalian cells. Here we have introduced photoproduct-specific DNA photolyases into a mouse cell line carrying the transgenic mutation reporter genes lacI and cII. Exposure of the photolyase-expressing cell lines to photoreactivating light resulted in almost complete repair of either CPDs or (6-4)PPs within less than 3 h. The mutations produced by the remaining, nonrepaired photoproducts were scored. The mutant frequency in the cII gene after photoreactivation by CPD photolyase was reduced from 127 x 10(-5) to 34 x 10(-5) (background, 8-10 x 10(-5)). Photoreactivation with (6-4) photolyase did not lower the mutant frequency appreciably. In the lacI gene the mutant frequency after photoreactivation repair of CPDs was reduced from 148 x 10(-5) to 28 x 10(-5) (background, 6-10 x 10(-5)). Mutation spectra obtained with and without photoreactivation by CPD photolyase indicated that the remaining mutations were derived from background mutations, unrepaired CPDs, and other DNA photopoducts including perhaps a small contribution from (6-4)PPs. We conclude that CPDs are responsible for at least 80% of the UVB-induced mutations in this mammalian cell model.     

Increased UV resistance of a xeroderma pigmentosum revertant cell line is correlated with selective repair of the transcribed strand of an expressed gene.

A UV-resistant revertant (XP129) of a xeroderma pigmentosum group A cell line has been reported to be totally deficient in repair of cyclobutane pyrimidine dimers (CPDs) but proficient in repair of 6-4 photoproducts. This finding has been interpreted to mean that CPDs play no role in cell killing by UV. We have analyzed the fine structure of repair of CPDs in the dihydrofolate reductase gene in the revertant. In this essential, active gene, we observe that repair of the transcribed strand is as efficient as that in normal, repair-proficient human cells, but repair of the nontranscribed strand is not. Within 4 h after UV at 7.5 J/m2, over 50% of the CPDs were removed, and by 8 h, 80% of the CPDs were removed. In contrast, there was essentially no removal from the nontranscribed strand even by 24 h. Our results demonstrate that overall repair measurements can be misleading, and they support the hypothesis that removal of CPDs from the transcribed strands of expressed genes is essential for UV resistance.