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.     

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.     

UV damage-specific DNA-binding protein in xeroderma pigmentosum complementation group E

The gel mobility shift assay method revealed a specifically ultraviolet (UV) damage recognizing, DNA-binding protein in nuclear extracts of normal human cells. The resulted DNA/protein complexes caused the two retarded mobility shifts. Four xeroderma pigmentosum complementation group E (XPE) fibroblast strains derived from unrelated Japanese families were not deficient in such a DNA damage recognition/binding protein because of the normal complex formation and gel mobility shifts, although we confirmed the reported lack of the protein in the European XPE (XP2RO and XP3RO) cells. Thus, the absence of this binding protein is not always commonly observed in all the XPE strains, and the partially repair-deficient and intermediately UV-hypersensitive phenotype of XPE cells are much similar whether or not they lack the protein.     

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.     

Chromosome and blood marker studies in families of patients affected by xeroderma pigmentosum and trichothiodystrophy

Chromosome and blood marker studies were performed in the families of 4 patients in which the association of 2 rare recessive Mendelian disorders, xeroderma pigmentosum (XP-D) and trichothiodystrophy (TTD), was present. Blood genotypes did not indicate any linkage with the pathologic condition, nor any segregation anomaly. Cytogenetic analysis using high-resolution banding techniques showed a normal karyotype both in the heterozygous and in the homozygous individuals. These findings lead us to exclude a cytologically detectable chromosome rearrangement, such as a microdeletion, as a possible cause of the association of XP-D and TTD in our patients.     

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.     

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.     

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.     

Increase of sister chromatid exchanges in excision repair deficient xeroderma pigmentosum

Summary The distribution of spontaneous sister chromatid exchanges (SCEs) was studied in PHA-stimulated lymphocytes from 15 patients affected by xeroderma pigmentosum (XP). The study of unscheduled DNA synthesis (UDS) in twelve of these patients showed that seven were deficient and five proficient. The number of SCEs in XP patient cells was higher than in those of 19 controls, and the distributions of SCEs per cell were significantly different. However, the results varied when XP patients were considered in relation to their UDS: the group of XP patients with proficient UDS did not differ, whereas the group of XP patients with deficient UDS was very significantly different from controls. The group not tested for UDS was similar to the deficient UDS group. The possible relationship between the increase of SCEs and the type of DNA repair defect is discussed.     

Low-level DNA exchanges in normal human and xeroderma pigmentosum cells after UV irradiation

We have used a T4 endonuclease V assay method for UV-induced pryrimidine dimers in cellular DNA in vivo to obtain evidence for recombinational DNA exchanges after UV irradiation of normal human and Xeroderma pigmentosum (XP) cells. Our data indicate that the endonuclease-sensitive sites in excision-defective XP cells are removed very slowly from the irradiated parental strands and appear concomitantly in daughter strands newly synthesized during post-UV incubation. In the defective XP cells, the extent of appearance of sensitive sites in daughter strands synthesized during a period of 24 h after 10 J/m² appears to be small, probably less than 15% of the initial number of sensitive sites detected in cellular parental strands. Demonstration of such exchanges between normal-density parental and 5-bromodeoxyuridine-labeled daughter strands by alkaline CsCl isopycnic centrifugation was unsuccessful. Further, the extent is much lower in normal human cell because of their efficiet excision repair of the dimers before and after exchanges than in the defective XP cells.     

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.     

XPC initiation codon mutation in xeroderma pigmentosum patients with and without neurological symptoms

Two unrelated xeroderma pigmentosum (XP) patients, with and without neurological abnormalities, respectively, had identical defects in the XPC DNA nucleotide excision repair (NER) gene. Patient XP21BE, a 27-year-old woman, had developmental delay and early onset of sensorineural hearing loss. In contrast, patient XP329BE, a 13-year-old boy, had a normal neurological examination. Both patients had marked lentiginous hyperpigmentation and multiple skin cancers at an early age. Their cultured fibroblasts showed similar hypersensitivity to killing by UV and reduced repair of DNA photoproducts. Cells from both patients had a homozygous c.2T>G mutation in the XPC gene which changed the ATG initiation codon to arginine (AGG). Both had low levels of XPC message and no detectable XPC protein on Western blotting. There was no functional XPC activity in both as revealed by the failure of localization of XPC and other NER proteins at the sites of UV-induced DNA damage in a sensitive in vivo immunofluorescence assay. XPC cDNA containing the initiation codon mutation was functionally inactive in a post-UV host cell reactivation (HCR) assay. Microsatellite markers flanking the XPC gene showed only a small region of identity ( approximately 30kBP), indicating that the patients were not closely related. Thus, the initiation codon mutation resulted in DNA repair deficiency in cells from both patients and greatly increased cancer susceptibility. The neurological abnormalities in patient XP21BE may be related to close consanguinity and simultaneous inheritance of other recessive genes or other gene modifying effects rather than the influence of XPC gene itself.     

Recognition of helical kinks by xeroderma pigmentosum group A protein triggers DNA excision repair

The function of human XPA protein, a key subunit of the nucleotide excision repair pathway, has been examined with site-directed substitutions in its putative DNA-binding cleft. After screening for repair activity in a host-cell reactivation assay, we analyzed mutants by comparing their affinities for different substrate architectures, including DNA junctions that provide a surrogate for distorted reaction intermediates, and by testing their ability to recruit the downstream endonuclease partner. Normal repair proficiency was retained when XPA mutations abolished only the simple interaction with linear DNA molecules. By contrast, results from a K141E K179E double mutant revealed that excision is crucially dependent on the assembly of XPA protein with a sharp bending angle in the DNA substrate. These findings show how an increased deformability of damaged sites, leading to helical kinks recognized by XPA, contributes to target selectivity in DNA repair.     

RecQ4 facilitates UV light-induced DNA damage repair through interaction with nucleotide excision repair factor xeroderma pigmentosum group A (XPA)

Mutations in the RECQL4 helicase gene have been linked to Rothmund-Thomson syndrome, which is characterized by genome instability, cancer susceptibility, and premature aging. To better define the cellular function of the RecQ4 protein, we investigated the subcellular localization of RecQ4 upon treatment of cells with different DNA-damaging agents including UV irradiation, 4-nitroquinoline 1-oxide, camptothecin, etoposide, hydroxyurea, and H(2)O(2). We found that RecQ4 formed discrete nuclear foci specifically in response to UV irradiation and 4-nitroquinoline 1-oxide. We demonstrated that functional RecQ4 was required for the efficient removal of UV lesions and could rescue UV sensitivity of RecQ4-deficient Rothmund-Thomson syndrome cells. Furthermore, UV treatment also resulted in the colocalization of the nuclear foci formed with RecQ4 and xeroderma pigmentosum group A in human cells. Consistently, RecQ4 could directly interact with xeroderma pigmentosum group A, and this interaction was stimulated by UV irradiation. By fractionating whole cell extracts into cytoplasmic, soluble nuclear, and chromatin-bound fractions, we observed that RecQ4 protein bound more tightly to chromatin upon UV irradiation. Taken together, our findings suggest a role of RecQ4 in the repair of UV-induced DNA damages in human cells.     

Centrin 2 stimulates nucleotide excision repair by interacting with xeroderma pigmentosum group C protein

Xeroderma pigmentosum group C (XPC) protein plays a key role in DNA damage recognition in global genome nucleotide excision repair (NER). The protein forms in vivo a heterotrimeric complex involving one of the two human homologs of Saccharomyces cerevisiae Rad23p and centrin 2, a centrosomal protein. Because centrin 2 is dispensable for the cell-free NER reaction, its role in NER has been unclear. Binding experiments with a series of truncated XPC proteins allowed the centrin 2 binding domain to be mapped to a presumed alpha-helical region near the C terminus, and three amino acid substitutions in this domain abrogated interaction with centrin 2. Human cell lines stably expressing the mutant XPC protein exhibited a significant reduction in global genome NER activity. Furthermore, centrin 2 enhanced the cell-free NER dual incision and damaged DNA binding activities of XPC, which likely require physical interaction between XPC and centrin 2. These results reveal a novel vital function for centrin 2 in NER, the potentiation of damage recognition by XPC.     

Human chromosome 9 can complement UV sensitivity of xeroderma pigmentosum group A cells

A single human chromosome derived from normal human fibroblasts and tagged with the G418 resistance gene was transferred into SV40-transformed xeroderma pigmentosum group A (XP-A) cells via microcell fusion. When chromosome 1 or 12 was transferred, UV sensitivity of microcell hybrid cells was not changed. By contrast, after transferring chromosome 9, 7 of 11 recipient clones were as UV-resistant as normal human cells. Four other clones were still as UV-sensitive as the parental XP-A cells. Southern hybridization analysis using a polymorphic probe, pEKZ19.3, which is homologous to a sequence of the D9S17 locus on chromosome 9, has confirmed that at least a part of normal human chromosome 9 was transferred into the recipient clones. However, amounts of UV-induced unscheduled DNA synthesis in the UV-resistant clones were only one-third of those in normal human cells. These results indicate that a gene on chromosome 9 can confer complementation of high UV sensitivity of XP-A cells although it is still possible that 2 or more genes might be involved in the defective-repair phenotypes of XP-A.