A seventh complementation group in excision-deficient xeroderma pigmentosum.

Cells from a xeroderma pigmentosum patient XP2BI who has reached 17 years of age with no keratoses or skin tumours constitute a new, 7th complementation group G. These cells exhibit a low residual level of excision repair, 2% of normal after a UV dose of 5 J/m2 and an impairment of post-replication repair characteristic of excision-defective XPs. They are also sensitive to the lethal effects of UV and defective in host-cell reactivation of UV-irradiated SV40 DNA.     

Biochemical heterogeneity in xeroderma pigmentosum complementation group E.

Cells from two patients with xeroderma pigmentosum complementation group E (XP-E) have been shown to lack an activity which binds specifically to UV-irradiated DNA (Chu and Chang, 1988). We investigated the occurrence of this binding activity in cell strains from nine additional, unrelated XP-E patients and found that all but one of these strains contained normal levels of the binding protein. Furthermore, the binding activity from these XP-E strains was indistinguishable from that of normal controls in thermal stability, behavior on ion-exchange chromatography, and electrophoretic mobility of protein-DNA complexes, indicating that there were no gross structural alterations in the protein. The association of XP-E with a deficiency in DNA-damage binding protein in cells from 3 of 12 XP-E patients (compared to 0 of 20 non-XP-E controls) is statistically significant (p less than 0.05), but there is no obvious correlation between the biochemical defect and the clinical or cellular characteristics of individual patients. Implications of these findings for the role of the binding protein in XP-E are discussed.     

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.     

A ninth complementation group in xeroderma pigmentosum, XP I

A new complementation group of excision-deficient xeroderma pigmentosum (XP) is described in 2 patients living in the F.R.G. Dermatological, ophthalmological and neurological symptoms of XP are presented together with DNA repair characteristics such as unscheduled DNA synthesis, colony-forming ability and alkaline elution studied in cultured fibroblasts. The results are compared to normal controls.     

Correlation between complementation group for immortality and DNA synthesis inhibitors

Previous studies had demonstrated that a DNA synthesis inhibitor(s) was produced by senescent but not young human diploid fibroblasts (HDF). Analysis of immortal human cell lines led to the finding that SUSM-1, carcinogen-treated immortal human liver fibroblast cells, expressed a potent inhibitor of DNA synthesis that was active in proliferation-competent young HDF but did not affect the SUSM-1 cell line itself. To determine whether one mechanism of escape from senescence to the immortal phenotype involved the loss of response to such DNA synthesis inhibitors, we initiated the present study analyzing a larger number of immortal human cell lines representative of the four complementation groups for indefinite division identified to date. We have found a correlation between the assignment of a cell line to Complementation Group D and the production of DNA synthesis inhibitors coupled with inability to respond to the inhibitory factors. We have also observed a correlation between the ability of immortal cell lines to respond to such DNA synthesis inhibitory factors and assignment to Complementation Group B. These data suggest DNA synthesis inhibitors are involved in the limited lifespan of normal cells and that the immortalization process may involve alterations in the activity of or response to such inhibitors.     

Cockayne syndrome complementation group B associated with xeroderma pigmentosum phenotype

Two siblings have been reported whose clinical manifestations (cutaneous photosensitivity and central nervous system dysfunction) are strongly reminiscent of the DeSanctis-Cacchione syndrome (DCS) variant of xeroderma pigmentosum (XP), a severe form of XP. Fibroblasts from the siblings showed UV sensitivity, a failure of recovery of RNA synthesis (RRS) after UV irradiation, and a normal level of unscheduled DNA synthesis (UDS), which were, unexpectedly, the biochemical characteristics usually associated with Cockayne syndrome (CS). However, no complementation group assignment in these cells has yet been performed. We here report that these patients can be assigned to CS complementation group B (CSB) by cell fusion complementation analysis. To our knowledge, these are the first patients with defects in the CSB gene to be associated with an XP phenotype. The results imply that the gene product from the CSB gene must interact with the gene products involved in excision repair and associated with XP.     

Xeroderma pigmentosum complementation group G associated with Cockayne syndrome.

Xeroderma pigmentosum (XP) and Cockayne syndrome (CS) are two rare inherited disorders with a clinical and cellular hypersensitivity to the UV component of the sunlight spectrum. Although the two traits are generally considered as clinically and genetically distinct entities, on the biochemical level a defect in the nucleotide excision-repair (NER) pathway is involved in both. Classical CS patients are primarily deficient in the preferential repair of DNA damage in actively transcribed genes, whereas in most XP patients the genetic defect affects both "preferential" and "overall" NER modalities. Here we report a genetic study of two unrelated, severely affected patients with the clinical characteristics of CS but with a biochemical defect typical of XP. By complementation analysis, using somatic cell fusion and nuclear microinjection of cloned repair genes, we assign these two patients to XP complementation group G, which previously was not associated with CS. This observation extends the earlier identification of two patients with a rare combined XP/CS phenotype within XP complementation groups B and D, respectively. It indicates that some mutations in at least three of the seven genes known to be involved in XP also can result in a picture of partial or even full-blown CS. We conclude that the syndromes XP and CS are biochemically closely related and may be part of a broader clinical disease spectrum. We suggest, as a possible molecular mechanism underlying this relation, that the XPGC repair gene has an additional vital function, as shown for some other NER genes.     

Temperature-sensitive defect of vesicular stomatitis virus in complementation group II.

The prototype member of the complementation group II temperature-sensitive (ts) mutants of vesicular stomatitis virus, ts II 052, has been investigated. In ts II 052-infected HeLa cells at the restrictive temperature (39.5 degrees C), reduced viral RNA synthesis was observed by comparison with infections conducted at the permissive temperature (30 degrees C). It was found that for an infection conducted at 39.5 degrees C, no 38S RNA or intracytoplasmic nucleocapsids were present. For nucleocapsids isolated from ts II 052 purified virions or from ts II 052-infected cells at 30 degrees C, the RNA was sensitive to pancreatic RNase after an exposure at 39.5 degrees C in contrast to the resistance observed for wild-type virus. The nucleocapsid stability of wild-type virus when heated to 63 degrees C or submitted to varying pH was not found in nucleocapsids extracted from ts II 052 purified virions. The data suggest that for ts II 052 there is an altered relationship between the viral 38S RNA and the nucleocapsid protein(s) by comparison with wild-type virus. Such results argue for the complementation group II gene product being N protein, so that the ts defect in ts II 052 represents an altered N protein.     

Xeroderma pigmentosum complementation group A protein acts as a processivity factor

We have previously shown that endonucleases present in a protein complex, which has specificity for cyclobutane pyrimidine dimers, locate sites of damage in DNA by a processive mechanism of action in normal human lymphoblastoid cells. In contrast, the endonucleases present in this complex from xeroderma pigmentosum complementation group A (XPA) cells locate damage sites by a distributive or significantly less processive mechanism. Since the XPA protein has been shown to be responsible for the DNA repair defect in XPA cells, this protein was examined for involvement in the mechanism of target site location of these endonucleases. A recombinant XPA protein, produced by expression of the normal XPA cDNA in E. coli, was isolated and purified. The results show that the recombinant XPA protein was able to correct the defect in ability of the XPA endonucleases to act by a processive mechanism of action on UVC irradiated DNA. These studies indicate that the XPA protein, in addition to a role in damage recognition or damage verification, may function as a processivity factor.     

Phenotype-genotype relationships in complementation group 3 of the peroxisome-biogenesis disorders.

The peroxisome-biogenesis disorders (PBDs) are a set of often lethal genetic diseases characterized by mental retardation and defective peroxisomal matrix protein import. Mutations in PEX12 are known to underlie the disease in two patients from complementation group 3 of the PBDs. Here we show that all patients from this group carry mutations on both alleles of PEX12. A comparison between PEX12 genotypes and the clinical and cellular phenotypes of the corresponding PBD patients suggests a relatively straightforward relationship between genotype and phenotype in this group of the PBDs, such that the loss of PEX12 function leads to more-severe cellular and clinical phenotypes. However, one patient who presented relatively mild clinical and cellular phenotypes was a compound heterozygote for two seemingly severe mutations on each PEX12 allele. PEX12 mRNA present in the patient's cells was derived from only one allele, the one that carried a 2-bp deletion early in the PEX12 coding region, c.26,27Delta. The deduced protein product of this mRNA would contain only the first eight amino acids of the protein, and yet this mutant PEX12 cDNA displayed significant PEX12 activity in a functional complementation assay. Surprisingly, the PEX12/c.26, 27Delta cDNA directed the synthesis of a 29-kD PEX12 protein in vitro, a result that is consistent with translation initiation at a downstream AUG codon. Transfection studies confirmed the expression of similarly sized PEX12 proteins from the PEX12/c.26,27Delta allele. Thus, it appears that translation initiation at internal AUG codons may modulate disease phenotypes and should be considered whenever unexpectedly mild phenotypes result from severe mutations early in the coding region.     

Intracellular localization of the Fanconi anemia complementation group A protein.

Mutations in the Fanconi anemia (FA) complementation group A (FANCA) gene leads to bone marrow failure, developmental abnormalities and cancer predisposition. To map the intracellular site of FANCA, we constructed a plasmid vector which linked in-frame the enhanced green fluorescent protein (EGFP cDNA) to the 5' end of the FANCA cDNA (pDAS-3). We studied the expression of pDAS-3 in the FANCA mutant fibroblast cell line (GM6914). MMC sensitivity of pDAS-3 transfected cells was comparable to wild-type fibroblasts. The resulting fluorescence pattern in the stable pDAS-3 cell line expressing the fusion protein was primarily nuclear. EGFP-selected cells (lacking FANCA) remain hypersensitive to MMC and maintained a cytoplasmic fluorescence pattern. Using deletion mutants of pDAS-3, a nuclear localization domain was identified at the amino terminus of the polypeptide. Western blot results of FANCA protein confirmed the presence of FANCA in nuclear fractions and FANCA protein levels did not vary during cell cycling. This nuclear trafficking of FANCA should guide future work in defining the function of this protein.     

Cell lines from xeroderma pigmentosum complementation group A lack a single-stranded-DNA-binding activity

Human fibroblasts and HeLa cells contain two major DNA-binding activities for superhelical DNA, which can be separated by phosphocellulose chromatography. The DNA-binding activity which elutes first from the column coelutes with and is probably identical to a single-stranded-DNA-binding activity. The second activity has been characterized previously. It binds preferentially to super-helical DNA containing DNA damage, but does not bind to single-stranded DNA. Five cell lines derived from patients with the repairdeficiency syndrome xeroderma pigmentosum (XP) were analyzed for the presence of these binding activities. Four of the cell lines were from the A-complementation group and one was from the D-complementation group of XP. The binding activity with preference for damaged DNA was present in all cell lines. The single-stranded-DNA-binding activity was present in the XP-D cell line but was absent or reduced in all of the four XP-A cell lines tested.     

Mitochondrial respiratory chain Complex I defects in Fanconi anemia complementation group A

Fanconi anemia (FA) is a rare and complex inherited blood disorder of the child. At least 15 genes are associated with the disease. The highest frequency of mutations belongs to groups A, C and G. Genetic instability and cytokine hypersensitivity support the selection of leukemic over non-leukemic stem cells.