UV-induced DNA repair synthesis in cells of patients with different forms of xeroderma pigmentosum and of heterozygotes

UV-induced DNA repair synthesis, as measured by autoradiography as well as by isopycnic centrifugation methods, was studied in a large number of cell strains from patients with the classic form of xeroderma pigmentosum (XP) or the De Sanctis-Cacchione syndrome (DSC) and several of their heterozygous parents. On the basis of the kinetics of repair synthesis in the cultured skin fibroblasts we have recognized four distinct groups of XP patients: (1) classic XP patients with low residual repair capacities, (2) classic XP patients with intermediate, but dose-dependent, levels of repair synthesis relative to the normal level, (3) patients, diagnosed as having classic XP, with a normal or only slightly reduced repair capacity and (4) DSC patients with a complete deficiency of repair synthesis. Complementation studies reported elsewhere have shown that different mutations are responsible for the detect in at least three of these groups. Cell strains of each of the four XP types were able to rejoin single-strand DNA breaks induced by X-rays. Most of the cell strains derived from heterozygotes showed normal repair activities. However, in the parents some of the of DSC-patients a significant reduction of the level of repair synthesis was found.     

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.     

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.     

Repair of DNA damage after exposure to 4-nitroquinoline-1-oxide in heterokaryons derived from xeroderma pigmentosum cells

Xeroderma pigmentosum (XP) cells are dificient in the repair of damage induced by ultraviolet irradiation. Excision-repair-deficient XP cell strains have been classified into 7 distinct complementation groups, according to results of studies on cell fusion and UV irradiation. XP cells are not only abnormally sensitive to UV, but also to a variety of chemical carcinogens, including 4-nitroquinoline-1-oxide (4NQO). Complementation analysis with XP strains from 4 different complementation groups with respect to the repair of 4NQO-induced DNA damage revealed that the classification of the strains into complementation groups with respect to 4NQO-induced repair coincides with the classification based on the repair of UV damage.     

Transient correction of excision repair defects in fibroblasts of 9 xeroderma pigmentosum complementation groups by microinjection of crude human cell extracts

Crude extracts from human cells were microinjected into the cytoplasm of cultured fibroblasts from 9 excision-deficient xeroderma pigmentosum (XP) complementation groups. The level of UV-induced unscheduled DNA synthesis (UDS) was measured to determine the effect of the extract on the repair capacity of the injected cells. With a sensitive UDS assay procedure a (transient) increase in UV-induced UDS level was found in fibroblasts from all complementation groups after injection of extracts from repair-proficient (HeLa) or complementing XP cells (except in the case of XP-G), but not after introduction of extracts from cells belonging to the same complementation group. This indicates that the phenotypic correction is exerted by complementation-group-specific factors in the extract, a conclusion that is in agreement with the observation that different levels of correction are found for different complementation groups. The XP-G-correcting factor was shown to be sensitive to proteolytic degradation, suggesting that it is a protein like the XP-A factor.     

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.     

Repair replication in heterokaryons deprived from different repair-deficient xeroderma pigmentosum strains

Repair replication was studied in UV-irradiated cell populations obtained after fusion of cell strains originating from different xeroderma pigmentosum (XP) patients. The capacity to perform repair replication appeared to be restored completely in multinucleate heterokaryons resulting from fusion between a classic XP-strain and a De Sanctis-Cacchione (DSC) strain. In cell populations obtained by fusion of either two different classic XP strains or two different DSC strains no repair replication was observed.These results, obtained with the technique of density labelling and isopycnic centrifugation of DNA, confirm our previously reported results of autoradiographic studies of unscheduled DNA synthesis. The occurrence of complementation between a classic XP strain and a DSC strain indicates that the defect in the two forms of the disease is caused by different mutations.     

Interaction of human and chick DNA repair functions in UV-irradiated xeroderma pigmentosum-chick erythrocyte heterokaryons

Fusion of chick erythrocytes with human primary fibroblasts results in the formation of heterokaryons in which the inactive chick nuclei become reactivated. The expression of chick DNA repair functions was investigated by the analysis of the DNA repair capacity after exposure to ultraviolet (UV) irradiation of such heterokaryons obtained after fusion of chick erythrocytes with normal human or xeroderma pigmentosum (XP) cells of complementation groups A, B, C and D. Unscheduled DNA synthesis (UDS) in normal human nuclei in these heterokaryons is suppressed during the first 2–4 days after fusion. The extent and duration of this suppression is positively correlated with the number of chick nuclei in the heterokaryons. Suppression is absent in heterokaryons obtained after fusion of chicken embryonic fibroblasts with XP cells (complementation group A and C).Restoration of DNA repair synthesis is found after fusion in XP nuclei of all complementation groups studied. It occurs rapidly in XP group A nuclei, starting one day after fusion and reaching near normal human levels after 5–8 days. In nuclei of the B, C and D group increased levels of UDS are found 5 days after fusion. At 8 days after fusion the UDS level is about 50% of that found in normal human nuclei. The pattern of UDS observed in the chick nuclei parallels that of the human counterpart in the fusion. A fast complementation pattern is also observed in chick fibroblast-XP group A heterokaryons resulting within 24 h in a UDS level comparable with that in chick fibroblast-normal human heterokaryons. In heterokaryons obtained after fusion of chick fibroblasts with XP group C cells UDS remains at the level of chick cells. These data suggest that reactivation of chick erythrocyte nuclei results in expression of repair functions which are able to complement the defects in the XP complementation groups A, B, C and D.     

Complementing xeroderma pigmentosum fibroblasts restore biological activity to UV-damaged DNA.

UV survival curves of adenovirus 2 using fused, complementing xeroderma pigmentosum (XP) fibroblast strains as virus hosts showed a component with an inactivation slope identical to that given by normal cells. This component was not observed when the fibroblasts were not fused or when fusion involved strains in the same complementation group. Extrapolation of this component indicated that at zero dose 3% of the viral plaque-forming units had infected cells capable of normal repair. These results suggest that 3% of the cells were complementing heterokaryons, a value similar to that actually observed by autoradiographic analysis of UV-induced unscheduled DNA synthesis. Thus, heterokaryons formed from XP fibroblasts belonging to different complementation groups are as capable of restoring biological activity to UV-damaged adenovirus 2 as are normal cells.     

Microinjection of human cell extracts corrects xeroderma pigmentosum defect

Cultured fibroblasts of patients with the DNA repair syndrome xeroderma pigmentosum (XP) were injected with crude cell extracts from various human cells. Injected fibroblasts were then assayed for unscheduled DNA synthesis (UDS) to see whether the injected extract could complement their deficiency in the removal of u.v.-induced thymidine dimers from their DNA. Microinjection of extracts from repair-proficient cells (such as HeLa, placenta) and from cells belonging to XP complementation group C resulted in a temporary correction of the DNA repair defect in XP-A cells but not in cells from complementation groups C, D or F. Extracts prepared from XP-A cells were unable to correct the XP-A repair defect. The UDS of phenotypically corrected XP-A cells is u.v.-specific and can reach the level of normal cells. The XP-A correcting factor was found to be sensitive to the action of proteinase K, suggesting that it is a protein. It is present in normal cells in high amounts, it is stable on storage and can still be detected in the injected cells 8 h after injection. The microinjection assay described in this paper provides a useful tool for the purification of the XP-A (and possibly other) factor(s) involved in DNA repair.     

Microinjection of Escherichia coli UvrA, B, C and D proteins into fibroblasts of xeroderma pigmentosum complementation groups A and C does not result in restoration of UV-induced unscheduled DNA synthesis

The UV-induced unscheduled DNA synthesis (UDS) in cultured human fibroblasts of repair-deficient xeroderma pigmentosum complementation groups A and C was assayed after injection of identical activities of either Uvr excinuclease (UvrA, B, C and D) from Escherichia coli or endonuclease V from phage T4. Under conditions where the T4 enzyme was able to induce repair synthesis in both XP complementation groups in agreement with earlier observations (de Jonge et al., 1985), no effect of the UvrABCD excinuclease could be observed either when the enzymatic complex was injected into the cytoplasm, or when it was delivered directly into the nucleus. In addition, no effect of the E. coli excinuclease was found on the repair ability of normal repair-proficient human fibroblasts. We conclude that the UvrABCD excinuclease may not work on DNA lesions in human chromatin.     

Infection of UV-irradiated xeroderma pigmentosum fibroblasts by herpes simplex virus: study of capacity and Weigle reactivation.

The capacity of monolayers of both normal human and xeroderma pigmentosum (XP) filbroblasts to support plaque formation by herpes simplex virus was decreased when the monolayers were ultraviolet (UV) irradiated and infected with virus. Fibroblasts of XP complementation groups A, B, and D were sensitive to UV, being 4-6 fold more sensitive than either fibroblasts of XP complementation group C or fibroblasts from a normal individual. When the monolayers were irradiated 4 days prior to infection, the capacity of normal fibroblasts to support herpes virus growth recovered, whereas the capacity of the XP strains decreased further compared to that measured when infection immediately followed irradiation. Concurrent experiments with UV-irradiated herpes virus showed that the survival of this virus did not increase when infection by irradiated virus immediately followed irradiation of the monolayers. However, if the monolayers were irradiated 4 days prior to infection, the survival of this virus increased by a factor of nearly 2. Such Weigle reactivation (WR) occurred at lower fluences to the XP fibroblasts than to normal fibroblasts, suggesting that WR results from residual cellular DNA damage left after excision repair.     

DDB2-Independent Role for p53 in the Recovery from Ultraviolet Light-Induced Replication Arrest

Ultraviolet light (UV light) induces helix distorting DNA lesions that pose a block to replicative DNA polymerases. Recovery from this replication arrest is reportedly impaired in nucleotide excision repair (NER)-deficient xeroderma pigmentosum (XP) fibroblasts and primary fibroblasts lacking functional p53. These independent observations suggested that the involvement of p53 in the recovery from UV-induced replication arrest was related to its role in regulating the global genomic subpathway of NER (GG-NER). Using primary human fibroblasts, we confirm that the recovery from UV-induced replication arrest is impaired in cells lacking functional p53 and in primary XP fibroblasts derived from complementation groups A or C (XP-A and XP-C) that are defective in GG-NER. Surprisingly, DNA synthesis recovered normally in GG-NER-deficient XP complementation group E (XP-E) cells that carry mutations in the p53 regulated DNA repair gene DDB2 and are specifically defective in the repair of cyclobutane pyrimidine dimers (CPD) but not pyrimidine (6-4) pyrimidone photoproducts. Disruption of p53 in these XP-E fibroblasts prevented the recovery from UV-induced replication arrest. Therefore, the roles of p53 and GG-NER in the recovery from UV-induced replication are separable and DDB2-independent. These results further indicate that primary human fibroblasts expressing functional p53 efficiently replicate DNA containing CPD whereas p53-deficient cells do not, consistent with a role for p53 in permitting translesion DNA synthesis of these DNA lesions.     

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.     

Differences in the levels of UV repair and in clinical symptoms in two sibs affected by xeroderma pigmentosum

Summary UV-repair activity was studied in two sibs affected by XP showing different clinical symptoms. Complementation studies indicated that both patients fit into complementation group A. The levels of UV-induced ³H-thymidine incorporation, in fibroblasts and in lymphocytes, are different in the two patients: residual level of repair DNA synthesis in the sister is higher than in the brother. In one of the cell samples analyzed UDS analysis showed that in the sister a low proportion of cells with normal repair synthesis is present.     

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.     

Isolation and partial characterization of virus-transformed cell lines representing the A, G and variant complementation groups of xeroderma pigmentosum

We have established viral-transformed, apparently permanent (immortalized) cell lines from diploid fibroblasts representative of normal and xeroderma pigmentosum (XP) A, G and variant individuals. The XP-G and XP-variant cells represent complementation groups not previously available as permanent lines. All the new permanent cell lines exhibit SV40 T-antigen expression. They are also aneuploid and have growth characteristics typical of viral transformants. They have retained the phenotypes of UV sensitivity, reduced repair synthesis or defective 'postreplication repair' appropriate to the XP complementation group they represent. Additionally, the new cell lines are all transfectable with the selectable plasmid pRSVneo. The XP-G and XP-variant cell lines show enhanced transfection with UV-irradiated plasmid DNA; a phenomenon previously reported for normal immortalized cells and for immortalized cells from the A and F complementation groups of XP.     

The endogenous nuclease sensitivity of repaired DNA in human fibroblasts

The limited DNA excision repair that occurs in the chromatin of UV-irradiated growth arrested cells isolated from a xeroderma pigmentosum (XP) complementation group C patient is clustered in localized regions. The repaired DNA was found to be more sensitive to nicking by endogenous nucleases than the bulk of the DNA. The extra-sensitivity does not change with increasing amounts of DNA damage or repair activity in the locally-repaired regions and is retained through a 24-h chase period. We suggest that these results are due to the occurrence of DNA repair limited to pre-existing, non-transient chromatin fractions that contain actively transcribed DNA. A similar extra-sensitivity of repaired DNA was not detected in cells of normal or XP complementation group A strains that exhibit either normal or limited repair located randomly throughout their genomes. The association between endogenous nuclease sensitivity and clustered repair probably defines a normal excision repair pathway that is specific for selected chromatin domains. The repair defect in XP-C strains may be one in pathways targeted for other endogenous nuclease-resistant domains.