Defective repair of gamma-ray induced DNA damage in xeroderma pigmentosum cells.

We used the bromouracil-photolysis technique to estimate the sizes of the repaired regions in normal human and xeroderma pigmentosum (XP) cells irradiated by gamma-rays aerobically or anoxically. After 1 1/2 hours of incubation, single-strand breaks were repaired and the repaired regions were small--one to two BrUra residues--for cells irradiated aerobically or anoxically. After a 20-hour incubation, the repaired region in normal cells showed a component mimicking U.V.-repair. There were large patches (approximately 30 BrUra residues) in the approximate ratios of one per six chain breaks for aerobic irradiation and one per three chain breaks for anoxic irradiation. XP cells, however, only showed large patches at 20 hours if they had been irradiated aerobically. We could not detect such regions in XP cells irradiated anoxically. These results indicate (1) that some part of ionizing damage mimics excision of U.V. damage in that the repair patches are large and the repair takes an appreciable time; (2) the types of such damage depend on whether the irradiation is done aerobically or anoxically; and (3) XP cells are defective in repairing a component of anoxic damage.     

Defective repair replication of DNA in xeroderma pigmentosum. 1968

Most forms of the human hereditary disease xeroderma pigmentation (XP) are due to a defect in nucleotide excision repair of DNA damage in skin cells associated with exposure to sunlight. This discovery by James Cleaver had an important impact on our understanding of nucleotide excision repair in mammals.     

Regulation of DNA repair in serum-stimulated xeroderma pigmentosum cells

The regulation of DNA repair during serum stimulation of quiescent cells was examined in normal human cells, in fibroblasts from three xeroderma pigmentosum complementation groups (A, C, and D), in xeroderma pigmentosum variant cells, and in ataxia telangiectasia cells. The regulation of nucleotide excision repair was examined by exposing cells to ultraviolet irradiation at discrete intervals after cell stimulation. Similarly, base excision repair was quantitated after exposure to methylmethane sulfonate. WI-38 normal human diploid fibroblasts, xeroderma pigmentosum variant cells, as well as ataxia telangiectasia cells enhanced their capacity for both nucleotide excision repair and for base excision repair prior to their enhancement of DNA synthesis. Further, in each cell strain, the base excision repair enzyme uracil DNA glycosylase was increased prior to the induction of DNA polymerase using the identical cells to quantitate each activity. In contrast, each of the three xeroderma complementation groups that were examined failed to increase their capacity for nucleotide excision repair above basal levels at any interval examined. This result was observed using either unscheduled DNA synthesis in the presence of 10 mM hydroxyurea or using repair replication in the absence of hydroxyurea to quantitate DNA repair. However, each of the three complementation groups normally regulated the enhancement of base excision repair after methylmethane sulfonate exposure and each induced the uracil DNA glycosylase prior to DNA synthesis. These results suggest that there may be a relationship between the sensitivity of xeroderma pigmentosum cells from each complementation group to specific DNA damaging agents and their inability to regulate nucleotide excision repair during cell stimulation.     

Defective DNA endonuclease activity on anthramycin treated DNA in xeroderma pigmentosum and mouse melanoma cells

Nine separate DNA endonuclease activities from non-histone chromatin proteins and a corresponding set from the nucleoplasm of normal human and xeroderma pigmentosum, complementation group A, lymphoblastoid and Cloudman mouse melanoma cells, obtained by isoelectric focusing, were tested against circular duplex phage PM2 DNA previously treated with anthramycin. A marked increase in activity against anthramycin treated DNA was found in normal human lymphoblastoid cells in a chromatin fraction with pI 4.6, with lesser increases at pI's 3.9 and 5.4 and a nucleoplasmic fraction at pI 4.6. In the nuclear proteins of xeroderma pigmentosum and mouse melanoma cells, however, no increase in activity against anthramycin DNA could be detected in any fraction.     

Mechanisms of impairment of DNA repair in human cells. Interferons stimulated DNA repair in xeroderma pigmentosum cells

DNA repair synthesis and strand break DNA repair induced by 4-nitroquinoline-1-oxide and UV-irradiation in Xeroderma pigmentosum lymphocytes and fibroblasts pretreated by leucocyte interferons were studied. Stimulation of DNA repair synthesis in interferon-pretreated Xeroderma pigmentosum cells, defective in incision, was detected. No such effect was noted for strand break DNA repair. Hence, antimutagenic activity of interferons in human cells is connected with their modificating effect on DNA repair.     

Defective recovery of semi-conservative DNA synthesis in xeroderma pigmentosum cells following split-dose ultraviolet irradiation.

In normal human fibroblasts we observe an enhancement of the recovery of the rate of semi-conservative DNA synthesis after split-dose UV-irradiation relative to a single total UV dose. The enhanced recovery is totally absent in both a xeroderma pigmentosum variant line and two xeroderma pigmentosum lines belonging to complementation groups A and C.     

Excision repair characteristics of denV-transformed xeroderma pigmentosum cells

Introduction of the denV gene of phage T4, encoding the pyrimidine dimer-specific endonuclease V, into xeroderma pigmentosum cells XP12RO(M1) was reported to result in partial restoration of colony-forming ability and excision repair synthesis. We have further characterized 3 denV-transformed XP clones in terms of rates of excision of pyrimidine dimers and size of the resulting resynthesized regions following exposure to 100 J/m2 from an FS-40 sunlamp. In the denV-transformed XP cells we observed 50% dimer removal within 3-6 h after UV exposure as compared to no measurable removal in the XP12RO(M1) line and 50% dimer excision after 18 h in the GM637A human, control cells. Dimer removal was assayed with Micrococcus luteus UV-endonuclease in conjunction with sedimentation of treated DNA in alkaline sucrose gradients. The size of the resulting repaired regions was determined by the bromouracil photolysis technique. Based on the photolytic sensitivity of DNA repaired in the presence of bromodeoxyuridine, we calculated that the excision of a dimer in the GM637A cells appears to be accompanied by the resynthesis of a region approximately 95 nucleotides in length. Conversely, the resynthesized regions in the denV-transformed clones were considerably smaller and were estimated to be between 13 and 18 nucleotides in length. These results may indicate that either the endonuclease that initiated dimer repair dictated the size of the resynthesized region or that the long-patch repair observed in the normal cells resulted from the repair of non-dimer DNA lesions.     

Complementation of the xeroderma pigmentosum DNA repair defect in cell-free extracts

Soluble extracts from human lymphoid cell lines that perform repair synthesis on covalently closed circular DNA containing pyrimidine dimers or psoralen adducts are described. Short patches of nucleotides are introduced by excision repair of damaged DNA in an ATP-dependent reaction. Extracts from xeroderma pigmentosum cell lines fail to act on damaged circular DNA, but are proficient in repair synthesis of ultraviolet-irradiated DNA containing incisions generated by Micrococcus luteus pyrimidine dimer-DNA glycosylase. Repair is defective in extracts from all xeroderma pigmentosum cell lines investigated, representing the genetic complementation groups A, B, C, D, H, and V. Mixing of cell extracts of group A and C origin leads to reconstitution of the DNA repair activity.     

DNA methylation in xeroderma pigmentosum

DNA methylation was examined in xeroderma pigmentosum (XP) cells. The amount of 5-methylcytosine (mC) in DNA from XP cells was about 70% of that in DNA from normal controls. Southern hybridization analysis showed that the HLA-DR alpha gene in XP lymphocyte B cells was differently methylated from normals, but its expression was apparently unaffected. The methylation of dihydrofolate reductase, a housekeeping gene, was the same as in controls. The revertants to UV resistance from XP fibroblasts recovered a methylation level close to that of normal cells. Results suggested that XP DNA was undermethylated non-randomly, and that DNA methylation might be associated with DNA repair function.     

Unique DNA repair properties of a xeroderma pigmentosum revertant.

A group A xeroderma pigmentosum revertant with normal sensitivity was created by chemical mutagenesis. It repaired (6-4) photoproducts normally but not pyrimidine dimers and had near normal levels of repair replication, sister chromatid exchange, and mutagenesis from UV light. The rate of UV-induced mutation in a shuttle vector, however, was as high as the rate in the parental xeroderma pigmentosum cell line.     

Fluorescent-light-induced lethality and DNA repair in normal and xeroderma pigmentosum fibroblasts

Cell survival and induction of endonuclease-sensitive sites in DNA were measured in human fibroblast cells exposed to fluorescent light or germicidal ultraviolet light. Cells from a xeroderma pigmentosum patient were hypersensitive to cell killing by fluorescent light, although less so than for germicidal ultraviolet light. Xeroderma pigmentosum cells were deficient in the removal of fluorescent light-induced endonuclease sites that are probably pyrimidine dimers, and both the xeroderma pigmentosum and normal cells removed these sites with kinetics indistinguishable from those for ultraviolet light-induced sites. A comparison of fluorescent with ultraviolet light data demonstrates that there are markedly fewer pyrimidine dimers per lethal event for fluorescent than for ultraviolet light, suggesting a major role for non-dimer damage in fluorescent light lethality.     

Defective repair of mitomycin C crosslinks in Fanconi's anemia and loss in confluent normal human and xeroderma pigmentosum cells

Crosslink repair of mitomycin C-induced interstrand crosslinks was studied in exponentially growing and confluent normal human, transformed WI38CT-1, Fanconi's anemia (FA) and xeroderma pigmentosum (XP) group-A fibroblasts by the assay methods of alkaline sucrose centrifugation, hydroxyapatite column chromatography and S₁-nuclease digestion. These three methods demonstrated unequivocally that crosslinking occurred at a rate of 0.13 crosslinks/10⁸ Da per μg per ml mitomycin C ( 10 μg/ml) and the first half-excision of crosslinks followed the rapid first-order kinetics of 2–3 h half-life in exponentially-growing normal, WI38CT-1 and XP group-A cells. However, the first half-excision was completely defective in three out of the four FA strains tested and severely retarded in an FA strain. These results strongly support our previous observations in different strains of normal human, FA and XP group-A cells. An important new addition is that confluent, otherwise proficient, normal and XP cells almost completely lost the ability of the first, rapid half-excision of mitomycin C crosslinks in their DNA. This probably suggests that the enzyme or regulatory factor responsible for the half-excision, which differs from that for nucleotide excision repair, present constitutively in confluent cells, may be induced or activated only in the cycling cells. However, its relation to a defective FA factor is not clear at present.     

Defective repair of mitomycin C crosslinks in Fanconi's anemia and loss in confluent normal human and xeroderma pigmentosum cells

Crosslink repair of mitomycin C-induced interstrand crosslinks was studied in exponentially growing and confluent normal human, transformed W138CT-1, Fanconi's anemia (FA) and xeroderma pigmentosum (XP) group-A fibroblasts by the assay methods of alkaline sucrose centrifugation, hydroxyapatite column chromatography and S1-nuclease digestion. These three methods demonstrated unequivocally that crosslinking occurred at a rate of 0.13 crosslinks/10(8) Da per microgram per ml mitomycin C (less than or equal to 10 micrograms/ml) and the first half-excision of crosslinks followed the rapid first-order kinetics of 2-3 h half-life in exponentially-growing normal, WI38CT-1 and XP group-A cells. However, the first half-excision was completely defective in three out of the four FA strains tested and severely retarded in an FA strain. These results strongly support our previous observations in different strains of normal human, FA and XP group-A cells. An important new addition is that confluent, otherwise proficient, normal and XP cells almost completely lost the ability of the first, rapid half-excision of mitomycin C crosslinks in their DNA. This probably suggests that the enzyme or regulatory factor responsible for the half-excision, which differs from that for nucleotide excision repair, present constitutively in confluent cells, may be induced or activated only in the cycling cells. However, its relation to a defective FA factor is not clear at present.     

DNA base changes and RNA levels in N-acetoxy-2-acetylaminofluorene-induced dihydrofolate reductase mutants of Chinese hamster ovary cells

Formerly, we isolated a series of dihydrofolate reductase-deficient Chinese hamster ovary cell mutants that were induced by N-acetoxy-2-acetylaminofluorene. Deletions and complex gene rearrangements were detected in 28% of these mutants; 72% contained putative point mutations. In the present study, we have localized the putative point mutations in the 25,000 base dhfr gene by RNase heteroduplex mapping. Assignment of a position for each mutation was successful in 16 of 19 mutants studied. We cloned DNA fragments containing the mapped mutations from nine mutants into a bacteriophage lambda vector. In the case of 11 other mutants, DNA was amplified by the polymerase chain reaction procedure. Sequence analysis of cloned and amplified DNA confirmed the presence of point mutations. Most mutants (90%) carried base substitutions; the rest contained frameshift mutations. Of the point mutations, 75% were G.C to T.A transversions in either the dhfr coding sequence or at splice sites; transition G.C to A.T mutations were found in two mutants (10%). In one of these transition mutants, the base substitution occurred at the fifth base of the third intron. Of the frameshift mutations, one was a deletion of G.C pair and the other was an insertion of an A.T pair. Of the mapped mutants, 38% exhibited greatly reduced (approximately 10-fold) steady-state levels of dhfr mRNA. All eight sequenced mutants displaying this phenotype contained premature chain termination codons. Normal levels of dhfr mRNA were observed in five missense mutants and in five mutants carrying nonsense codons in the translated portion of exon VI. Taken together with the results of other mutagens at this locus, we conclude that the low dhfr mRNA phenotype is correlated with the presence of nonsense codons in exons II to V but not in the last exon of the dhfr gene.     

Repair of gamma-ray-induced DNA base damage in xeroderma pigmentosum cells

The repair of DNA damage produced by 137Cs gamma irradiation was measured with a preparation from Micrococcus luteus containing DNA damage-specific endonucleases in combination with alkaline elution. The frequency of these endonuclease sensitive sites (ESS) was determined after 54 or 110 Gy of oxic irradiation in normal and xeroderma pigmentosum (XP) fibroblasts from complementation groups A, C, D, and G. Repair was rapid in all cell strains with greater than 50% repair after 1.5 h of repair incubation. At later repair times, 12-17 h, more ESS remained in XP than in normal cells. The frequency of excess ESS in XP cells was approximately 0.04 per 10(9) Da of DNA per Gy which was equivalent to 10% of the initial ESS produced. The removal of ESS was comparable in XP cells with normal radiosensitivity and XP3BR cells which have been reported to be moderately radiosensitive.     

Dose-dependent increase in repair of 1-beta-D-arabinofuranosylcytosine-detectable DNA lesions in UV-treated xeroderma pigmentosum (group A) fibroblasts

The extent of DNA-excision repair was determined in human fibroblast strains from clinically normal and xeroderma pigmentosum complementation group A (XP-A) donors after irradiation with 254-nm ultraviolet (UV) light. Repair was monitored by the use of 1-beta-D-arabinofuranosylcytosine (araC), a potent inhibitor of DNA synthesis, and alkaline sucrose velocity sedimentation to quantitate DNA single-strand breaks. In this approach, the number of araC-accumulated breaks in post-UV incubated cultures becomes a measure of the efficiency of a particular strain to perform long-patch excision repair. The maximal rate of removal of araC-detectable DNA lesions equalled approximately 1.8 sites/10(8) dalton/h in the normal strains (GM38, GM43), while it was more than 10-fold lower in both XP-A strains (XP4LO, XP12BE) examined. In normal fibroblasts the number of lesions removed during the first 4 h after irradiation saturated at approximately 10 J/m2. In contrast, the residual amount of repair in the excision-deficient cells increased as a linear function of UV fluence over a range 5-120 J/m2. Thus we conclude that the repair of araC-detectable UV photoproducts in XP group A fibroblasts is limited by availability of damaged regions in the genome to repair complexes.     

Retrovirus-mediated DNA repair gene transfer into xeroderma pigmentosum cells: Perspectives for a gene therapy

The rare hereditary disease xeroderma pigmentosum (XP) is clinically characterized by extreme sun sensitivity and an increased predisposition for developing skin cancer. Cultured cells from XP patients exhibit hypersensitivity to ultraviolet (UV) radiation due to the defect in nucleotide excision repair (NER), and other cellular abnormalities. Seven genes identified in the classical XP forms, XPA to XPG, are involved in the NER pathway. In view of developing a strategy of gene therapy for XP, we devised recombinant retrovirus-carrying DNA repair genes for transfer and stable expression of these genes in cells from XP patients. Results showed that these retroviruses are efficient tools for transducing XP fibroblasts and correcting repair-defective cellular phenotypes by recovering normal UV survival, unscheduled DNA synthesis, and RNA synthesis after UV irradiation, and also other cellular abnormalities resulting from NER defects. These results imply that the first step of cellular gene therapy might be accomplished successfully.