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.     

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.     

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.     

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.     

Correction of the ultraviolet light induced DNA-repair defect in xeroderma pigmentosum cells by electroporation of a normal human endonuclease

Cells from patients with xeroderma pigmentosum, complementation group A (XPA), are known to be defective in repair of pyrimidine dimers and other forms of damage produced by 254-nm ultraviolet (UVC) radiation. We have isolated a DNA endonuclease, pI 7.6, from the chromatin of normal human lymphoblastoid cells which recognizes damage produced by UVC light, and have introduced this endonuclease into UVC-irradiated XPA cells in culture to determine whether it can restore their markedly deficient DNA repair-related unscheduled DNA synthesis (UDS). Introduction of the normal endonuclease, which recognizes predominantly pyrimidine dimers, but not the corresponding XPA endonuclease into UVC-irradiated XPA cells restored their levels of UDS to approximately 80% of normal values. Electroporation of both the normal and the XPA endonuclease into normal human cells increases UDS in normal cells to higher than normal values. These results indicate that the normal endonuclease can restore UDS in UVC-irradiated XPA cells. They also indicate that XPA cells have an endonuclease capable of increasing the efficiency of repair of UVC damage in normal cells.     

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.     

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.     

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.     

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.