Correction of the DNA repair defect in xeroderma pigmentosum group E by injection of a DNA damage-binding protein.

Cells from a subset of patients with the DNA-repair-defective disease xeroderma pigmentosum complementation group E (XP-E) are known to lack a DNA damage-binding (DDB) activity. Purified human DDB protein was injected into XP-E cells to test whether the DNA-repair defect in these cells is caused by a defect in DDB activity. Injected DDB protein stimulated DNA repair to normal levels in those strains that lack the DDB activity but did not stimulate repair in cells from other xeroderma pigmentosum groups or in XP-E cells that contain the activity. These results provide direct evidence that defective DDB activity causes the repair defect in a subset of XP-E patients, which in turn establishes a role for this activity in nucleotide-excision repair in vivo.     

Microcell-mediated transfer of a single human chromosome complements xeroderma pigmentosum group A fibroblasts.

Chromosomes from an immortalized aneuploid human fibroblast cell line were randomly tagged with the selectable marker neo by transfection with the plasmid pSV2neo. Somatic cell fusions between transfected human cells and mouse A9 cells generated pools of G418-resistant human-mouse hybrid clones containing various numbers of human chromosomes. Microcell-mediated chromosome transfer from the hybrid pools to xeroderma pigmentosum complementation group A (XP-A) cells in culture and selection for G418-resistant colonies resulted in the identification of XP cells with enhanced resistance to ultraviolet radiation. Screening of subclones from selected pools of human-mouse hybrids facilitated the identification of hybrids containing a single neo-tagged human chromosome. Transfer of this chromosome to XP-A cells (but not to XP-F or XP-C cells) results in enhanced resistance to ultraviolet light and enhanced excision repair capacity. The identification of a single human chromosome that complements the phenotype of XP-A cells in culture provides the potential for genetic mapping of the complementing gene and for its isolation by molecular cloning.     

Molecular cloning of a mouse DNA repair gene that complements the defect of group-A xeroderma pigmentosum.

For isolation of the gene responsible for xeroderma pigmentosum (XP) complementation group A, plasmid pSV2gpt and genomic DNA from a mouse embryo were cotransfected into XP2OSSV cells, a group-A XP cell line. Two primary UV-resistant XP transfectants were isolated from about 1.6 X 10(5) pSV2gpt-transformed XP colonies. pSV2gpt and genomic DNA from the primary transfectants were again cotransfected into XP2OSSV cells and a secondary UV-resistant XP transfectant was obtained by screening about 4.8 X 10(5) pSV2gpt-transformed XP colonies. The secondary transfectant retained fewer mouse repetitive sequences. A mouse gene that complements the defect of XP2OSSV cells was cloned into an EMBL3 vector from the genome of a secondary transfectant. Transfections of the cloned DNA also conferred UV resistance on another group-A XP cell line but not on XP cell lines of group C, D, F, or G. Northern blot analysis of poly(A)+ RNA with a subfragment of cloned mouse DNA repair gene as the probe revealed that an approximately 1.0 kilobase mRNA was transcribed in the donor mouse embryo and secondary transfectant, and approximately 1.0- and approximately 1.3-kilobase mRNAs were transcribed in normal human cells, but none of these mRNAs was detected in three strains of group-A XP cells. These results suggest that the cloned DNA repair gene is specific for group-A XP and may be the mouse homologue of the group-A XP human gene.     

Correction of xeroderma pigmentosum complementation group D mutant cell phenotypes by chromosome and gene transfer: involvement of the human ERCC2 DNA repair gene.

Cultured cells from individuals afflicted with the genetically heterogeneous autosomal recessive disorder xeroderma pigmentosum (XP) exhibit sensitivity to UV radiation and defective nucleotide excision repair. Complementation of these mutant phenotypes after the introduction of single human chromosomes from repair-proficient cells into XP cells has provided a means of mapping the genes involved in this disease. We now report the phenotypic correction of XP cells from genetic complementation group D (XP-D) by a single human chromosome designated Tneo. Detailed molecular characterization of Tneo revealed a rearranged structure involving human chromosomes 16 and 19, including the excision repair cross-complementing 2 (ERCC2) gene from the previously described human DNA repair gene cluster at 19q13.2-q13.3. Direct transfer of a cosmid bearing the ERCC2 gene conferred UV resistance to XP-D cells.     

Stable low molecular weight DNA in xeroderma pigmentosum cells.

Xeroderma pigmentosum (XP) cells from several complementation groups contained more low molecular weight DNA upon alkaline sucrose gradient centrifugation than did other human cells examined. Under conditions in which only 5% of the DNA in normal cells sedimented at 16 S or less, 20% of the DNA in XP cells from complementation group A sedimented at 16 S or less. Because cells were layered directly onto the gradients for lysing of cells and denaturing of the DNA, it appears that this low molecular weight material is due to naturally existing gaps or alkali-sensitive sites, or both, in the cellular DNA. The increase in low molecular weight DNA seen in XP complementation group A cells also is seen in complementation groups C, D, and E. When prelabeled cells were incubated for increasing times after removal of the radioactive label, the amount of low molecular weight material remained constant over a 3-hr period. The introduction of the DNA-damaging agent, bleomycin, to prelabeled XP cells produced a surprising effect. The normal response of human cells to bleomycin is an increase in low molecular weight DNA, dependent on the dose of the drug and time of treatment. In XP cells the reverse was observed. That is, the low molecular weight DNA observed in untreated XP cells disappeared upon addition of the drug. The process responsible for the unusual response of XP cells to bleomycin is unknown, but these results are compatible with an inducible repair process.     

DNA single-strand breaks during repair of UV damage in human fibroblasts and abnormalities of repair in xeroderma pigmentosum.

The method of DNA alkaline elution was applied to a study of the formation and resealing of DNA single-strand breaks after irradiation of human fibroblasts with ultraviolet light (UV). The general features of the results were consistent with current concepts of DNA excision repair, in that breaks appeared rapidly after UV, and resealed slowly in normal fibroblasts, whereas breaks did not appear in those cells of patients with xeroderma pigmentosum (XP) that are known to have defects in DNA repair synthesis. The appearance of breaks required a short post-UV incubation, consistent with the expected action of an endonuclease. Cells of the variant form of XP characterized by normal DNA repair synthesis exhibited normal production of breaks after UV, but were slower than normal cells in resealing these breaks. This difference was enhanced by caffeine. A model is proposed to relate this finding with a previously described defect in post-replication repair in these XP variant cells. DNA crosslinking appears to cause an underestimate in the measurement of DNA breakage after UV.     

Complementation of the UV-sensitive phenotype of a xeroderma pigmentosum human cell line by transfection with a cDNA clone library.

In previous work, a xeroderma pigmentosum cell line belonging to complementation group C was established by transformation with origin-defective simian virus 40. We now report the complementation of the UV sensitivity of this cell line by gene transfer. A human cDNA clone library constructed in a mammalian expression vector, and itself incorporated in a lambda phage vector, was introduced into the cells as a calcium phosphate precipitate. Following selection to G418 resistance, provided by the neo gene of the vector, transformants were selected for UV resistance. Twenty-one cell clones were obtained with UV-resistance levels typical of normal human fibroblasts. All transformants contained vector DNA sequences in their nuclei. Upon further propagation in the absence of selection for G418 resistance, about half of the primary transformants remained UV-resistant. Secondary transformants were generated by transfection with a partial digest of total chromosomal DNA from one of these stable transformants. This resulted in 15 G418-resistant clones, 2 of which exhibited a UV-resistant phenotype. The other primary clones lost UV resistance rapidly when subcultured in the absence of G418. Importantly, several retained UV resistance under G418 selection pressure. The acquisition of UV resistance by secondary transformants derived by transfection of DNA from a stable primary transformant, and the linkage between G418 and UV resistances in the unstable primary transformants, strongly suggests that the transformants acquired UV resistance through DNA-mediated gene transfer and not by reversion.     

Increased UV resistance in xeroderma pigmentosum group A cells after transformation with a human genomic DNA clone.

Xeroderma pigmentosum (XP) is an autosomal recessive disease in which the major clinical manifestation is a 2,000-fold enhanced probability of developing sunlight-induced skin tumors, and the molecular basis for the disease is a defective DNA excision repair system. To clone the gene defective in XP complementation group A (XP-A), cDNA clones were isolated by a competition hybridization strategy in which the corresponding mRNAs were more abundant in cells of the obligately heterozygous parents relative to cells of the homozygous proband affected with the disease. In this report, a human genomic DNA clone that contains this cDNA was transformed into two independent homozygous XP-A cell lines, and these transformants displayed partial restoration of resistance to the killing effects of UV irradiation. The abundance of mRNA corresponding to this cDNA appears to correlate well with the observed UV cell survival. The results of unscheduled DNA synthesis after UV exposure indicate that the transformed cells are repair proficient relative to that of the control XP-A cells. However, using this same genomic DNA, transformation of an XP-F cell line did not confer any enhancement of UV survival or promote unscheduled DNA synthesis after UV exposure.     

Xeroderma pigmentosum cells with normal levels of excision repair have a defect in DNA synthesis after UV-irradiation.

Cells cultured from most patients suffering from the sunlight-sensitive hereditary disorder xeroderma pigmentosum are defective in the ability to excise ultraviolet light (UV)-induced pyrimidine dimers from their DNA. There is, however, one class of these patients whose cells are completely normal in this excision repair process. We have found that these cells have an abnormality in the manner in which DNA is synthesized after UV-irradiation. The time taken to convert initially low-molecular-weight DNA synthesized in UV-irradiated cells into high-molecular-weight DNA similar in size to that in untreated cells is much greater in these variants than in normal cells. Furthermore, this slow conversion of low to high-molecular-weight newly synthesized DNA is drastically inhibited by caffeine, which has no effect in normal cells. Two cell lines from classes of xeroderma pigmentosum that are defective in excision-repair show intermediate effects, with regard to both the time taken to convert newly synthesized DNA to high molecular weight and the inhibition of this process by caffeine.     

Human chromosome 13 compensates a DNA repair defect in UV-sensitive mouse cells by mouse--human cell hybridization.

A human chromosome responsible for excision repair of UV-induced DNA damage has been identified by studying somatic cell hybrids between an UV-sensitive mutant of mouse lymphoma L5178Y cells and normal human lymphocytes. An autosomal recessive mutant, Q31, of complementation group I is deficient in excision repair of UV-induced DNA damage. Somatic cell hybrids between Q31 and human lymphocytes exhibited the same UV resistance as did parental L5178Y cells. In addition, both the levels of UV-induced unscheduled DNA synthesis and chromosomal sensitivity were recovered from the UV-resistant hybrid clones. Segregation of the hybrid cells gave rise to UV-sensitive clones. The segregation of UV sensitivity was not correlated with the loss of human X chromosome. Karyotype analysis of the segregants gave evidence that a gene on human chromosome 13 compensates for UV hypersensitivity of Q31 mutant.     

DNA excision-repair defect of xeroderma pigmentosum prevents removal of a class of oxygen free radical-induced base lesions.

Plasmid DNA was gamma-irradiated or treated with H2O2 in the presence of Cu2+ to generate oxygen free radical-induced lesions. Open circular DNA molecules were removed by ethidium bromide/CsCl density gradient centrifugation. The closed circular DNA fraction was treated with the Escherichia coli reagent enzymes endonuclease III (Nth protein) and Fpg protein. This treatment converted DNA molecules containing the major base lesions pyrimidine hydrates and 8-hydroxyguanine to a nicked form. Remaining closed circular DNA containing other oxygen radical-induced base lesions was used as a substrate for nucleotide excision-repair in a cell-free system. Extracts from normal human cells, but not extracts from xeroderma pigmentosum cells, catalyzed repair synthesis in this DNA. The repair defect in the latter extracts could be specifically corrected by in vitro complementation. The data suggest that accumulation of endogenous oxidative damage in cellular DNA from xeroderma pigmentosum patients contributes to the increased frequency of internal cancers and the neural degeneration occurring in serious cases of the syndrome.     

Phage T4 endonuclease V stimulates DNA repair replication in isolated nuclei from ultraviolet-irradiated human cells, including xeroderma pigmentosum fibroblasts

The repair mode of DNA replication has been demonstrated in isolated nuclei from UV-irradiated human cells. Nuclei are incubated in a mixture containing [(3)H]thymidine triphosphate and bromodeoxyuridine triphosphate in a 1:5 ratio. The (3)H at the density of parental DNA in alkaline CsCl density gradients is then a measure of repair. In nuclei prepared from WI38 cells 30 min after irradiation, repair replication is UV dependent and proceeds at approximately the in vivo rate for 5 min. Repair replication is reduced in irradiated nuclei or in nuclei prepared immediately after irradiation. It is Mg(2+)-dependent and stimulated by added ATP and deoxyribonucleoside triphosphates. No repair replication is observed in nuclei from xeroderma pigmentosum (complementation group A) cells. However, upon addition of coliphage T4 endonuclease V, which specifically nicks DNA containing pyrimidine dimers, repair replication is observed in nuclei from irradiated xeroderma pigmentosum cells and is stimulated in WI38 nuclei. The reaction then persists for an hour and is dependent upon added ATP and deoxyribonucleoside triphosphates. The repair label is in stretches of roughly 35 nucleotides, as it is in intact cells. Added pancreatic DNase does not promote UV-dependent repair synthesis. Our results support the view that xeroderma pigmentosum (group A) cells are defective in the incision step of the DNA excision repair pathway, and demonstrate the utility of this system for probing DNA repair mechanisms.     

DNA切除修复基因XPD在肝癌中遗传易感性的荟萃分析

目的 探讨DNA切除修复基因XPD基因多态性在中国人群原发性肝癌中的遗传易感性. 方法 检索中外数据库,获得有关XPD基因多态性与肝癌发病风险的病例对照研究资料进行Meta分析,得到合并的优势比(OR)和95％可信区间(95％CI). 结果 共纳入XPD基因多态位点相关文献6篇,累计病例3424例,对照3636例;在XPD基因多态位点751和312位点等位基因的OR (95％CI)分别为1.25 (0.70～ 2.24)和0.85 (0.58～ 1.25);在XPD基因多态位点751,与野生基因型Lys/Lys相比,(Lys/Gln+Gln/Gln)合并的OR(95％CI)为1.31(0.71 ～ 2.42);在XPD基因多态位点312位点,与野生基因型Asp/Asp相比,(Asp/Asn+Asn/Asn)合并的OR值(95％CI)为1.19 (0.73～ 1.95). 结论 XPD多态性遗传位点751和312不是中国人群原发性肝癌发病的风险因素.     

Defective thymine dimer excision by cell-free extracts of xeroderma pigmentosum cells.

Crude extracts of normal human diploid fibroblasts and of human peripheral blood lymphocytes excise thymine dimers from purified ultraviolet-irradiated DNA, or from the DNA presumably present as chromatin in unfractionated cell-free preparations of cells that had been labeled with [3H]thymidine. Extracts of xeroderma pigmentosum cells from complementation groups A, C, and D also excise thymine dimers from purified DNA, but extracts of group A cells do not excise dimers from the DNA of radioactively labeled unfractionated cell-free preparations.     

Specific action of T4 endonuclease V on damaged DNA in xeroderma pigmentosum cells in vivo.

The specific action of T4 endonuclease V on damaged DNA in xeroderma pigmentosum cells was examined using an in vivo assay system with hemagglutinating virus of Japan (Sendai virus) inactivated by UV light. A clear dose response was observed between the level of UV-induce unscheduled DNA synthesis of xeroderma pigmentosum cells and the amount of T4 endonuclease V activity added. The T4 enzyme was unstable in human cells, and its half-life was 3 hr. Fractions derived from an extract of Escherichia coli infected with T4V1, a mutant defective in the endonuclease V gene, showed no ability to restore the UV-induced unscheduled DNA synthesis of xeroderma pigmentosum cells. However, fractions derived from an extract of T4D-infected E. coli with endonuclease V activity were effective. The T4 enzyme was effective in xeroderma pigmentosum cells on DNA damaged by UV light but not in cells damaged by 4-nitroquinoline 1-oxide. The results of these experiments show that the T4 enzyme has a specific action on human cell DNA in vivo. Treatment with the T4 enzyme increased the survival of group A xeroderma pigmentosum cells after UV irradiation.     

Restricted ultraviolet mutational spectrum in a shuttle vector propagated in xeroderma pigmentosum cells.

A shuttle vector plasmid, pZ189, carrying a bacterial suppressor tRNA marker gene, was treated with ultraviolet radiation and propagated in cultured skin cells from a patient with the skin-cancer-prone, DNA repair-deficient disease xeroderma pigmentosum and in repair-proficient cells. After replication in the human cells, progeny plasmids were purified. Plasmid survival and mutations inactivating the marker gene were scored by transforming an indicator strain of Escherichia coli carrying a suppressible amber mutation in the beta-galactosidase gene. Plasmid survival in the xeroderma pigmentosum cells was less than that of pZ189 harvested from repair-proficient human cells. The point-mutation frequency in the 150-base-pair tRNA marker gene increased up to 100-fold with ultraviolet dose. Sequence analysis of 150 mutant plasmids revealed that mutations were infrequent at potential thymine-thymine dimer sites. Ninety-three percent of the mutant plasmids from the xeroderma pigmentosum cells showed G X C----A X T transitions, compared to 73% in the normal cells (P less than 0.002). There were significantly fewer transversions (P less than 0.002) (especially G X C----T X A) and multiple base substitutions (P less than 0.00001) than when pZ189 was passaged in repair-proficient cells. The subset of mutational changes that are common to ultraviolet-treated plasmids propagated in both repair-proficient and xeroderma pigmentosum skin cells may be associated with the development of ultraviolet-induced skin cancer in humans.     

Mutagenesis by third-strand-directed psoralen adducts in repair-deficient human cells: high frequency and altered spectrum in a xeroderma pigmentosum variant.

Psoralen-conjugated triple-helix-forming oligonucleotides have been used to generate site-specific mutations within mammalian cells. To investigate factors influencing the efficiency of oligonucleotide-mediated gene targeting, the processing of third-strand-directed psoralen adducts was compared in normal and repair-deficient human cells. An unusually high mutation frequency and an altered mutation pattern were seen in xeroderma pigmentosum variant (XPV) cells compared with normal, xeroderma pigmentosum group A (XPA), and Fanconi anemia cells. In XPV, targeted mutations were produced in the supF reporter gene carried in a simian virus 40 vector at a frequency of 30%, 3-fold above that in normal or Fanconi anemia cells and 6-fold above that in XPA. The mutations generated by targeted psoralen crosslinks and monoadducts in the XPV cells formed a pattern distinct from that in the other three cell lines, with mutations occurring not just at the damaged site but also at adjacent base pairs. Hence, the XPV cells may have an abnormality in trans-lesion bypass synthesis during repair and/or replication, implicating a DNA polymerase or an accessory factor as a basis of the defect in XPV. These results may help to elucidate the repair deficiency in XPV, and they raise the possibility that genetic manipulation via triplex-targeted mutagenesis may be enhanced by modulation of the XPV-associated activity in normal cells.     

Microinjection of partially purified protein factor restores DNA damage specifically in group A of xeroderma pigmentosum cells.

Microinjection of cell extracts prepared from both human placenta and HeLa cells into xeroderma pigmentosum (XP) cells of complementation group A restores unscheduled DNA synthesis (UDS) in these cells after UV irradiation [de Jonge, A., Vermeulen, W., Klein, B. & Hoeijmakers, J. (1983) EMBO J. 2, 637-641]. These cells also showed normal resistance to UV irradiation. The half-life of the factors in the cell extracts corresponding to the UDS activity (factor A) was 14 hr in XP cells of group A, and the maximal level of UDS was exerted 2 hr after microinjection. The factors were sensitive to protease treatment but not to RNase treatment and were found to be approximately equal to 160 and approximately equal to 90 kDa by gel filtration. These two fractions of the factor(s) acted specifically in XP cells of complementation group A among complementation groups A, B, C, D, F, G, and probably E and H.