A further definition of characteristics of DNA-excision repair in xeroderma pigmentosum complementation group A strains

The location in the genome of excision repair following exposure to UV (254 nm) of two XP complementation group A strains, XP12BE and XP8LO, that differ considerably in their excision-repair rates, have been determined. Capacity for repair in XP8LO has also been determined. Sites repaired in DNA in a 24-h post-UV period were located relative to the remaining pyrimidine dimers using the M. luteus UV-endonuclease to nick partially repaired DNA and sedimentation in alkaline sucrose to size the resulting DNA. Repair in group A occurs randomly throughout the genome in a manner similar to that observed for normal cells but in contrast to domain-limited repair in group C strains. This observation defines a further similarity of the excision repair detected in group A compared to normal cells that is in addition to the previously reported related characteristics of the respective excision rate curves. A reduced repair capacity in XP8LO relative to normal cells was detected. This strain, which repairs DNA at an initial rate identical to that of normal strains when irradiated with doses of 5 J/m2 or less, repairs DNA at a slower than normal but constant rate at higher doses. This leads to the suggestion that XP8LO is defective in the number of repair enzyme complexes compared to normal cells.     

The oxidative DNA lesion 8,5'-(S)-cyclo-2'-deoxyadenosine is repaired by the nucleotide excision repair pathway and blocks gene expression in mammalian cells

Xeroderma pigmentosum (XP) patients with inherited defects in nucleotide excision repair (NER) are unable to excise from their DNA bulky photoproducts induced by UV radiation and therefore develop accelerated actinic damage, including cancer, on sun-exposed tissue. Some XP patients also develop a characteristic neurodegeneration believed to result from their inability to repair neuronal DNA damaged by endogenous metabolites since the harmful UV radiation in sunlight does not reach neurons. Free radicals, which are abundant in neurons, induce DNA lesions that, if unrepaired, might cause the XP neurodegeneration. Searching for such a lesion, we developed a synthesis for 8,5'-(S)-cyclo-2'-deoxyadenosine (cyclo-dA), a free radical-induced bulky lesion, and incorporated it into DNA to test its repair in mammalian cell extracts and living cells. Using extracts of normal and mutant Chinese hamster ovary (CHO) cells to test for NER and adult rat brain extracts to test for base excision repair, we found that cyclo-dA is repaired by NER and not by base excision repair. We measured host cell reactivation, which reflects a cell's capacity for NER, by transfecting CHO and XP cells with DNA constructs containing a single cyclo-dA or a cyclobutane thymine dimer at a specific site on the transcribed strand of a luciferase reporter gene. We found that, like the cyclobutane thymine dimer, cyclo-dA is a strong block to gene expression in CHO and human cells. Cyclo-dA was repaired extremely poorly in NER-deficient CHO cells and in cells from patients in XP complementation group A with neurodegeneration. Based on these findings, we propose that cyclo-dA is a candidate for an endogenous DNA lesion that might contribute to neurodegeneration in XP.     

Repair of damage by ultraviolet radiation in xeroderma pigmentosum cell strains of complementation groups E and F

The xeroderma pigmentosum fibroblast strains XP2RO, complementation group E, and XP23OS, group F, were compared with normal human primary fibroblasts with regard to repair of damage induced by 254-nm UV. In XP2RO cells, repair DNA synthesis, measured by autoradiography (unscheduled DNA synthesis = UDS), was about 50% of the value found in normal human cells. In these cells also the removal of UV-induced sites recognized by a specific UV-endonuclease proceeds at a reduced rate. By having BUdR incorporated into the repaired regions, followed by the induction of breaks in these patches by 313-nm UV, it was shown that the reduced repair synthesis is not caused by a shorter length of the repair regions in XP2RO, but is solely due to a reduction in the number of sites removed by excision repair. In XP23OS a discrepancy was observed between the level of UDS, which was about 10% of the normal value, and other repair-dependent properties such as UV survival, host-cell reactivation and removal of UV-endonuclease-susceptible sites, which were less reduced than could be expected from the UDS level. However, when UDS was followed over a longer period than the 2 or 3 h normally used in UDS analysis, it appeared that in XP23OS cells, the rate of UDS remained constant whereas the rate decreased in normal control cells. Consequently, the residual level of UDS varies with the period over which it is studied.     

DNA repair in human xeroderma pigmentosum group C cells involves a different distribution of damaged sites in confluent and growing cells.

Xeroderma pigmentosum is a human disease consisting of several complementation groups that are deficient in excision repair. Group C is one in which excision repair occurs at about 20-30% of normal levels. The distribution of mended sites in relation to unrepaired sites has been determined by cutting remaining unrepaired pyrimidine dimers with Microccocus luteus UV endonuclease. The mended sites have been found clustered together in a fashion that depended on cell proliferation. In confluent group C cells, the mended sites were clustered in regions where dimer excision was as efficient as excision in the DNA of normal cells. In proliferating group C cells, however, mended sites were randomly dispersed. The total amount of repair replication was the same in confluent and proliferating cells. Since previous work has shown that confluent group C cells show more extensive recovery from the lethal effects of UV irradiation than some other groups, clustered repair may correlate with a more efficient mechanism of restoring cell viability. The different distribution of repaired sites during DNA replication may be the result of changes in the state of the substrate for repair or changes in the metabolic priorities of DNA polymerases.     

DNA repair in man: regulation by a multigene family and association with human disease

The major mechanism of repair of damage to DNA involves a conceptually simple process of enzymatic excision and resynthesis of small regions of DNA. In man and other mammals, this process is regulated by several gene loci; up to 15 mutually complementary genes or gene products may be involved. Repair deficiency results in an array of clinical symptoms in skin, central nervous system, and hematopoietic and immune systems, the major example being xeroderma pigmentosum (XP), a disease with a high incidence of cancer. Cloning repair genes by straightforward methods has proved difficult, but we have begun the effort by demonstrating that correction of a human repair deficiency can be achieved by transferring very small fragments of DNA from normal hamsters into XP cells. One of the complementation groups of XP cells (group C) appears to express a change in gene regulation such that these cells repair only a small clustered region of the DNA with high efficiency.     

A re-examination of the intragenome distribution of repaired sites in proliferating xeroderma pigmentosum complementation group C fibroblasts.

We find that rapidly proliferating fibroblasts from xeroderma pigmentosum complementation group C (XP-C) patients, cells that have a small residual DNA excision repair capacity, repair DNA in localized regions of the genome in a clustered pattern rather than at single sites in dispersed locations. This finding is similar to that observed earlier for nondividing cells but is in contrast to published results that indicate that the residual repair in proliferating XP-C cells is dispersed throughout the genome in a non-clustered pattern. While we detect the same amount of repair in both proliferating and nondividing cells, we also observe no shift from the clustered pattern of repair to a more dispersive pattern when nondividing cells are stimulated to proliferate by fresh serum addition. We have no obvious explanation for these discrepancies with the published results. We have noted previously that proliferating XP-C cells are very UV sensitive relative to normal cells while nondividing cells that exhibit the same amount of repair activity are relatively UV resistant. There is no satisfactory explanation for this change in relative response to the lethal effects of UV, a change not observed for cell strains from other XP complementation groups. However, we argue that clustered repair in specific genomic regions promotes survival in nondividing XP-C cells but does not promote survival in proliferating cells.     

A presumed DNA helicase encoded by ERCC-3 is involved in the human repair disorders xeroderma pigmentosum and Cockayne's syndrome

The human gene ERCC-3 specifically corrects the defect in an early step of the DNA excision repair pathway of UV-sensitive rodent mutants of complementation group 3. The predicted 782 amino acid ERCC-3 protein harbors putative nucleotide, chromatin, and helix-turn-helix DNA binding domains and seven consecutive motifs conserved between two superfamilies of DNA and RNA helicases, strongly suggesting that it is a DNA repair helicase. ERCC-3-deficient rodent mutants phenotypically resemble the human repair syndrome xeroderma pigmentosum (XP). ERCC-3 specifically corrects the excision defect in one of the eight XP complementation groups, XP-B. The sole XP-B patient presents an exceptional conjunction of two rare repair disorders: XP and Cockayne's syndrome. This patient's DNA contains a C----A transversion in the splice acceptor sequence of the last intron of the only ERCC-3 allele that is detectably expressed, leading to a 4 bp insertion in the mRNA and an inactivating frameshift in the C-terminus of the protein. Because XP is associated with predisposition to skin cancer, ERCC-3 can be considered a tumor-preventing gene.     

Dose-dependent increase in repair of 1-β-d-arabinofuranosylcytosinedetectable 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-β-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 ∼ 1.8 sites/10⁸ 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 ∼ 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.     

Caffeine inhibition of the repair of ultraviolet-irradiated adenovirus in human cells.

Caffeine is shown to block repair of ultraviolet-irradiated adenovirus 2 when the irradiated virus infects normal human fibroblasts from a xeroderma pigmentosum (XP) variant. Such blockage is not observed when the irradiated virus infects XP fibroblasts belonging to XP complementation group A. Thus normal and XP variant cells have a caffeine-sensitive repair process. This may be either excision or an excision dependent repair process because fibroblasts belonging to XP complementation group A are believed to lack the excision repair process.     

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.     

Excision repair in xeroderma pigmentosum group C but not group D is clustered in a small fraction of the total genome

DNA repair in xeroderma pigmentosum complementation groups C and D occurs at a low level. Measurements of pyrimidine dimers remaining in bulk DNA from the whole genome indicated very little excision in either complementation group. The repair sites in group C cells were, however, clustered together in small regions of the genome which appeared to be mended nearly as efficiently as the whole genome is mended in normal cells, while repair in group D cells was randomly distributed. Growth of normal cells in cycloheximide or 3-aminobenzamide neither inhibited repair nor altered the distribution of repair sites. Growth of normal cells in novobiocin or aphidicolin inhibited excision but repair remained randomly distributed. On the basis of these observations, and consideration of other cellular features of group C and D, we suggest that group C may represent a mutation which results in a low level of repair enzymes with normal function. Group D, on the other hand, may represent a mutation resulting in functionally defective repair enzymes.     

Repair of 4-nitroquinoline-1-oxide-induced DNA damage in normal human cells and cells from classical and variant xeroderma pigmentosum

The effect of 4-nitroquinoline-1-oxide (4NQO) upon 3 fibroblast cell lines derived from normal and xeroderma pigmentosum subjects have been compared. Excision-deficient XP cells (XP2BI), complementation group G, are nearly 200-fold more sensitive than normal cells to the lethal effect of 4NQO while XP variants (XP7TA), are 2-fold more sensitive. This cytotoxicity correlates with the levels of unscheduled DNA synthesis performed by the 3 cell lines. 4NQO causes a dose-related inhibition of DNA replication in all cell lines. However, newly replicated DNA synthesised immediately after treatment of cells with 4NQO is slightly smaller in XP7TA variant cells than in normal cells receiving the same dose of 4NQO, but DNA fragments in excision-deficient XP2BI are 50% smaller. It is likely that replicon elongation and joining together of newly replicated DNA fragments is dependent upon the excision of certain 4NQO-induced lesions, possibly normally repaired by a 'short-patch' repair process defective in XP2BI.     

The rate of removal of pyrimidine dimers in quiescent cultures of normal human and xeroderma pigmentosum cells

The rate of removal of pyrimidine dimers from DNA of UV (254 nm)-irradiated (1 J/m2) normal and xeroderma pigmentosum (XP) cells maintained in culture as nondividing populations was determined. Several normal and XP strains from complementation groups A, C and D were studied. The excision rates and survival ability of nondividing cells were examined to determine if an abnormal sensitivity was associated with a decreased rate of dimer excision. The results show that all normal strains studied excise pyrimidine dimers at the same rate, with the rate curve characterized by two components. All 'excision-deficient' XP strains excise dimers at a slower-than-normal rate, with the rate curves also characterized by two components. The rate constants for the first components of all of the XP strains (group A, C and D) are the same, one tenth of the normal rate constant, except for XP8LO (group A). XP8LO has a first-component rate constant similar to that of normal strains and a second component rate constant similar to that of other group A strains (XP12BE, XP25RO). Thus, the slower rate of dimer excision in XP8LO is due to a defect in the mechanism responsible for the second component of the excision-rate curve. In general, an abnormal sensitivity of nondividing cells to UV is associated with a reduced dimer-excision rate. A notable exception to this is the group C strain XP1BE which has an initial repair rate similar to that of group A XP12BE but is considerably more resistant when survival is measured.     

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.     

Phenotypic correction of the defect in xeroderma pigmentosum cells after fusion with isolated cytoplasts

The cybridization technique was used to study the role of cytoplasmic and nuclear factors in complementation of the repair defects in xeroderma pigmentosum (XP) cells. Cybrids were prepared by fusion of UV-exposed XP cells with cytoplasts derived from normal human or complementing XP cells. Phenotypic correction of the DNA repair defect measured by unscheduled DNA synthesis (UDS) occurred in these cybrids. The results show that the correcting factors are present in the cytoplasts and can move into the nucleus of the UV-exposed XP cell almost immediately after fusion. The defective repair in the nuclei of XP complementation group A cell strains is corrected with fast kinetics reaching normal UDS levels within 2 h after fusion. In the A-group cybrids the correcting activity decreased with a half-time of about 12 h. Correction of the XP group C defect occurred at a much slower rate, indicating that different factors are involved in the correction of the XP-A and XP-C defects.     

Complementation analysis of xeroderma pigmentosum variants

DNA repair after UV exposure was studied in multinucleate cells, obtained after fusion of excision-defective and variant xeroderma pigmentosum fibroblasts. Optimal fusion conditions were determined, facilitating the measurement of DNA replication in heterokaryons. In unirradiated multikaryons, entry into the S phase was depressed, when compared with unfused cells. The extent of the depression of S phase entry was dependent on the fusion conditions. In heterokaryons obtained after fusion of XP variant (6 different strains) with excision-defective XP (three cell strains from complementation groups A, C and D) both unscheduled DNA synthesis and postreplication repair after UV irradiation were restored to normal levels. In contrast, complementation was not observed after pairwise fusion of the XP variant cell strains. These results suggest that the XP variants comprise a single complementation group, different from complementation groups A, C and D.     

Repair of UV-endonuclease-susceptible sites in the 7 complementation groups of xeroderma pigmentosum A through G

7 strains of human primary fibroblasts were chosen from the complementation groups A through G of xeroderma pigmentosum; these strains are UV-sensitive and deficient in excision repair of UV damage on the criterion of unscheduled DNA synthesis (UDS). They were compared with normal human fibroblasts and one xeroderma pigmentosum variant with regard to their capacity to remove pyrimidine dimers, induced in their DNA by UV at 253.7 nm. The XP variant showed a normal level of dimer removal, whereas 6 of the other XP strains had a greatly reduced capacity to remove this DNA damage, in agreement with their individual levels of UDS. Strain XP230S (complementation group F), however, only showed a 20% reduction in the removal of dimers, which is much less than expected from the low level of UDS in this strain.