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Xeroderma pigmentosum variant (XP-V) cells are deficient in their ability to synthesize intact daughter DNA strands after UV irradiation. This deficiency results from mutations in the gene encoding DNA polymerase η, which is required for effecting translesion synthesis (TLS) past UV photoproducts. We have developed a simple cellular procedure to identify XP-V cell strains, and have subsequently analyzed the mutations in 21 patients with XP-V. The 16 mutations that we have identified fall into three categories. Many of them result in severe truncations of the protein and are effectively null alleles. However, we have also identified five missense mutations located in the conserved catalytic domain of the protein. Extracts of cells falling into these two categories are defective in the ability to carry out TLS past sites of DNA damage. Three mutations cause truncations at the C terminus such that the catalytic domains are intact, and extracts from these cells are able to carry out TLS. From our previous work, however, we anticipate that protein in these cells will not be localized in the nucleus nor will it be relocalized into replication foci during DNA replication. The spectrum of both missense and truncating mutations is markedly skewed toward the N-terminal half of the protein. Two of the missense mutations are predicted to affect the interaction with DNA, the others are likely to disrupt the three-dimensional structure of the protein. There is a wide variability in clinical features among patients, which is not obviously related to the site or type of mutation.

Xeroderma pigmentosum (XP)/Cockayne syndrome (CS) complex is a combination of clinical features of two rare genetic disorders in one individual. A sun-sensitive boy (XP20BE) who had severe symptoms of CS, with dwarfism, microcephaly, retinal degeneration, and mental impairment, had XP-type pigmentation and died at 6 y with marked cachexia (weight 14.5 lb) without skin cancers. We evaluated his cultured cells for characteristic CS or XP DNA-repair abnormalities. The level of ultraviolet (UV)-induced unscheduled DNA synthesis was less than 5% of normal, characteristic of the excision-repair defect of XP. Cell fusion studies indicated that his cells were in XP complementation group G. His cells were hypersensitive to killing by UV, and their post-UV recovery of RNA synthesis was abnormally low, features of both CS and XP. Post-UV survival of plasmid pSP189 in his cells was markedly reduced, and post-UV plasmid mutation frequency was higher than with normal cells, as in both CS and XP. Sequence analysis of the mutated plasmid marker gene showed normal frequency of plasmids with multiple base substitutions, as in CS, and an abnormally increased frequency of G:C-->A:T mutations, a feature of XP. Transfection of UV-treated pRSVcat with or without photoreactivation revealed that his cells, like XP cells, could not repair either cyclobutane pyrimidine dimers or non-dimer photoproducts. These results indicate that the DNA-repair features of the XP20BE (XP-G/CS) cells are phenotypically more like XP cells than CS cells, whereas clinically the CS phenotype is more prominent than XP.

The RAD51 gene of Saccharomyces cerevisiae encodes a RecA like protein, which is involved in the recombinational repair of double strand breaks. We have isolated the RAD51 homologue, rhp51+, of the distantly related yeast strain Schizosaccharomyces pombe by heterologous hybridization. DNA sequence analysis of the rhp51+ gene revealed an open reading frame of 365 amino acids. Comparison of the amino acid sequences of RAD51 and rhp51+ showed a high level of conservation: 69% identical amino acids. There are two Mlul sites in the upstream region which may be associated with cell cycle regulation of the rhp51+ gene. The rhp51+ null allele, constructed by disruption of the coding region, is extremely sensitive to X-rays, indicating that the rhp51+ gene, like RAD51, is also involved in the repair of X-ray damage. The structural and functional homology between rhp51+ and RAD51 suggests evolutionary conservation of certain steps in the recombinational repair pathway.

Complementation of the repair defect in hamster xrs mutants has been achieved by transfer of human chromosome 2 using the method of microcell-mediated chromosome transfer. The xrs mutants belong to ionizing radiation complementation group 5, are highly sensitive to ionizing radiation, and have an impaired ability to rejoin radiation-induced DNA double-strand breaks. Both phenotypes were corrected by chromosome 2, although the correction of radiation sensitivity was only partial. Complementation was achieved in two members of this complementation group, xrs6 and XR-V15B, derived independently from the CHO and V79 cell lines, respectively. The presence of human chromosome 2 in complemented clones was examined cytogenetically and by PCR analysis with primers directed at a human-specific long interspersed repetitive sequence or chromosome 2-specific genes. Complementation was observed in 25/27 hybrids, one of which contained only the q arm of chromosome 2. The two noncomplementing hybrids were missing segments of chromosome 2. The use of a back-selection system enabled the isolation of clones that had lost the human chromosome and these regained radiation sensitivity. Transfer of several other human chromosomes did not result in complementation of the repair defect in XR-V15B. These data show that the gene defective in xrs cells, XRCC5, which is involved in double-strand break rejoining, is located on human chromosome 2q.

Trichothiodystrophy is a genetic disease which in the majority of cases studied is associated with a deficiency in the ability to repair UV damage in cellular DNA. Three categories of UV response have been identified. In type 1 the response is completely normal, whereas type 2 cells are deficient in excision-repair, with properties indistinguishable from those of XP complementation group D. Type 3 cells have normal survival following UV-irradiation and normal rates of removal of cyclobutane pyrimidine dimer sites. Nevertheless repair synthesis is reduced by 50% in these cell strains and this is associated with a marked reduction in the repair of 6-4 photoproducts from cellular DNA. The present results show that 50% or more of repair synthesis at early times after irradiation of normal primary human fibroblasts is attributable to repair of 6-4 products. They also suggest that repair of cyclobutane dimers is crucial for cell survival.

The transcription factor TFIIH is involved in both basal transcription and DNA repair. Mutations in the XPD helicase component of TFIIH can result in the diverse clinical features associated with xeroderma pigmentosum (XP) and trichothiodystrophy (TTD). It is generally believed that the multi-system abnormalities associated with TTD are the result of a subtle deficiency in basal transcription. However, to date, there has been no clear demonstration of a defect in expression of any specific gene in individuals with these syndromes. Here we show that the specific mutations in XPD that cause TTD result in reduced expression of the beta-globin genes in these individuals. Eleven TTD patients with characterized mutations in the XPD gene have the haematological features of beta-thalassaemia trait, and reduced levels of beta-globin synthesis and beta-globin mRNA. All these parameters were normal in three patients with XP. These findings provide the first evidence for reduced expression of a specific gene in TTD. They support the hypothesis that many of the clinical features of TTD result from inadequate expression of a diverse set of highly expressed genes.

DNA polymerase η carries out translesion synthesis past UV photoproducts and is deficient in xeroderma pigmentosum (XP) variants. We report that polη is mostly localized uniformly in the nucleus but is associated with replication foci during S phase. Following treatment of cells with UV irradiation or carcinogens, it accumulates at replication foci stalled at DNA damage. The C-terminal third of polη is not required for polymerase activity. However, the C-terminal 70 aa are needed for nuclear localization and a further 50 aa for relocalization into foci. Polη truncations lacking these domains fail to correct the defects in XP-variant cells. Furthermore, we have identified mutations in two XP variant patients that leave the polymerase motifs intact but cause loss of the localization domains.

Ten DNA repair (rad) genes from the fission yeast, Schizosaccharomyces pombe were mapped to the 17 NotI fragments of the three chromosomes. Nine of the genes map to chromosome I, but there is no evidence for significant clustering.

The Saccharomyces cerevisiae RAD30 gene encodes a novel eukaryotic DNA polymerase, pol eta that is able to replicate across cis-syn cyclobutane pyrimidine dimers both accurately and efficiently. Very recently, a human homolog of RAD30 was identified, mutations in which result in the sunlight-sensitive, cancer-prone, Xeroderma pigmentosum variant group phenotype. We report here the cloning and localization of a second human homolog of RAD30. Interestingly, RAD30B is localized on chromosome 18q21.1 in a region that is often implicated in the etiology of many human cancers. The mouse homolog (Rad30b) is located on chromosome 18E2. The human RAD30B and mouse Rad30b mRNA transcripts, like many repair proteins, are highly expressed in the testis. In situ hybridization analysis indicates that expression of mouse Rad30b occurs predominantly in postmeiotic round spermatids. Database searches revealed genomic and EST sequences from other eukaryotes such as Aspergillus nidulans, Schizosaccharomyces pombe, Brugia malayi, Caenorhabditis elegans, Trypanosoma cruzi, Arabidopsis thaliana, and Drosophila melanogaster that also encode putative homologs of RAD30, thereby suggesting that Rad30-dependent translesion DNA synthesis is conserved within the eukaryotic kingdom.

Summary Xeroderma pigmentosum (XP) complementation group D is a heterogeneous group, containing patients with XP alone, rare cases with both XP and Cockayne syndrome, and patients with trichothiodystrophy (TTD). TTD is a rare autosomal recessive multisystem disorder associated, in many patients, with a defect in nucleotide-excision repair; but in contrast to XP patients, TTD patients are not cancer prone. In most of the repair-deficient TTD patients, the defect has been assigned to the XPD gene. The XPD gene product is a subunit of transcription factor TFIIH, which is involved in both DNA repair and transcription. We have determined the mutations and the pattern of inheritance of the XPD alleles in the 11 cases identified in Italy so far, in which the hair abnormalities diagnostic for TTD are associated with different disease severity but similar cellular photosensitivity. We have identified eight causative mutations, of which four have not been described before, either in TTD or XP cases, supporting the hypothesis that the mutations responsible for TTD are different from those found in other pathological phenotypes. Arg112his was the most common alteration in the Italian patients, of whom five were homozygotes and two were heterozygotes, for this mutation. The presence of a specifically mutated XPD allele, irrespective of its homozygous, hemizygous, or heterozygous condition, was always associated with the same degree of cellular UV hypersensitivity. Surprisingly, however, the severity of the clinical symptoms did not correlate with the magnitude of the DNA-repair defect. The most severe clinical features were found in patients who appear to be functionally hemizygous for the mutated allele.

The xeroderma pigmentosum group D (XPD) protein has a dual function, both in nucleotide excision repair of DNA damage and in basal transcription. Mutations in the XPD gene can result in three distinct clinical phenotypes, XP, trichothiodystrophy (TTD), and XP with Cockayne syndrome. To determine if the clinical phenotypes of XP and TTD can be attributed to the sites of the mutations, we have identified the mutations in a large group of TTD and XP-D patients. Most sites of mutations differed between XP and TTD, but there are three sites at which the same mutation is found in XP and TTD patients. Since the corresponding patients were all compound heterozygotes with different mutations in the two alleles, the alleles were tested separately in a yeast complementation assay. The mutations which are found in both XP and TTD patients behaved as null alleles, suggesting that the disease phenotype was determined by the other allele. If we eliminate the null mutations, the remaining mutagenic pattern is consistent with the site of the mutation determining the phenotype.

DNA repair defects in the xeroderma pigmentosum (XP) group D complementation group can be associated with the clinical features of two quite different disorders; XP, a sun-sensitive and cancer-prone disorder, or trichothiodystrophy (TTD) which is characterized by sulphur-deficient brittle hair and a variety of other associated abnormalities, but no skin cancer. The XPD gene product, a DNA helicase, is required for nucleotide excision repair and recent evidence has demonstrated a role in transcription. We have now identified causative mutations in XPD in four TTD patients. The patients are all compound heterozygotes and the locations of the mutations enable us to suggest relationships between different domains in the gene and its roles in excision repair and transcription.

Accurate dosimetry for chemical mutagens is extremely difficult, and precise manipulation of the frequency of a particular lesion is ordinarily impossible. With 8-MOP plus UVA, however, both are possible because 8-MOP, when photoactivated by one photon of UVA, forms monoadducts whilst crosslinks are formed only if a second photon of light photoactivates the monoadducts. If 8-MOP molecules that are unreacted after a UVA exposure are removed from cells by washing, the effect of a subsequent UVA irradiation can be attributed only to the conversion of monoadducts to DNA interstrand crosslinks. Using this experimental procedure and L5178Y mouse lymphoma cells, we have shown that DNA interstrand crosslinks are at least 10-fold more effective at causing both sister-chromatid exchanges and chromosomal aberrations than are monoadducts. In contrast, crosslinks are no more effective than monoadducts in mutation induction. These experiments identify directly for the first time that a particular chemically induced lesion, DNA interstrand crosslinks, can, like thymine dimers, cause chromosomal aberrations and sister-chromatid exchanges. The results also show that sister-chromatid exchanges can be induced independently of mutations.

46BR is a fibroblast cell strain established from an individual with hypogammaglobulinaemia. The cells unique in showing hypersensitivity to the lethal effects of a wide range of DNA-damaging agents. Thus they are hypersensitive to γ- and 254-nm UV-irradiation and show a limited capacity to repair potentially lethal γ-irradiation damage when compared with fibroblasts from normal individuals. A slight hypersensitivity to mitomycin C was also revealed but we were not able to discriminate 46BR from normals with 4-nitroquinoline oxide. The cells were hypersensitive to the alkylating agents, dimethyl sulphate, methyl methanesulphonate, ethyl methanesulphonate, N -methyl- N ′-nitro- N -nitrosoguanidine and N -methyl- N -nitrosourea but not N -ethyl- N -nitrosourea. A consideration of the spectra of DNA lesions produced by these alkylating agents together with the sensitivity to ionising radiation and mitomycin C suggests that 46BR cells are defective in a repair step that is common to all agents. We suggest that the cells are defective in DNA polymerisation or ligation. Support for this suggestion comes from the absence of any hypersensitivity to N -ethyl- N -nitrosourea since its major reaction products are not removed by excision pathways that require polymerisation and ligation.

The demonstration of a mutagenic activity in photocopy processes (1) and its identification and removal as an impurity in carbon black from some photocopying toners (2) is an excellent example of scientific detective work. the activity resides in nitropyrenes which are present in carbon blacks manufactured under some conditions. studies with various salmonella strains (2) showed that nitropyrenes were particularly potent frame-shift mutagens and that they may either be direct acting or require activation by an enzymatic pathway not involving the classical nitroreductase. the use of a standard S9 microsome fraction substantially reduced the mutagenic potency of a set of nitropyrenes.

• A uniform response to UV of four normal cell strains was demonstrated. One excision-proficient xeroderma pigmentosum variant strain (XP7TA) had a wild-type UV response but a second (XP30RO) was more sensitive. An excision-deficient xeroderma pigmentosum strain XP4LO was substantially more sensitive than wild-type cell strains. • A continuous post-irradiation treatment with non-toxic levels of caffeine enhanced the lethal effect of UV light in both xeroderma pigmentosum variant cell strains but not in cells from normal individuals. There was no detectable effect on cells from a xeroderma pigmentosum individual from complementation group A. These results correlate well with observations on the influence of caffeine on post-replication repair in the three classes of cells.

A fibroblast cell strain, 46BR, derived from an immunodeficient patient is hypersensitive to the lethal effects of a wide range of DNA-damaging agents. It is also defective in strand-break rejoining after treatment with dimethyl sulfate and UV light. The present study shows that the cells have a defect in joining Okazaki-type fragments during DNA replication, supporting the interpretation that the basic defect is in ligation of DNA strands. The baseline level of sister chromatid exchange is slightly higher than in normal cells but it does not approach that of Bloom's syndrome or dyskeratosis congenita cells. Sensitivity to the induction of sister chromatid exchange and the hypersensitivity to the lethal effects of a set of DNA-damaging agents are correlated, implying that the basic defect influences both end points in a similar manner. No 6-thioguanine-resistant mutants could be induced by either gamma- or UV-irradiation in these cells, suggesting that error-prone repair pathways for damage induced by these agents may contain a common ligation step in human cells.

The xeroderma pigmentosum group D (XPD) protein is a subunit of transcription factor TFIIH with DNA helicase activity. TFIIH has two functions, in basal transcription and nucleotide excision repair. Mutations in XPD that affect DNA repair but not transcription result in the skin cancer-prone disorder, xeroderma pigmentosum (XP). If transcription is also affected, the result is the multi-system disorder trichothiodystrophy (TTD), in which there is no skin cancer predisposition, or in rare cases, XP combined with Cockayne syndrome. Up till now there have been no reports of combined clinical features of XP and TTD. We have now identified two patients with some features of both these disorders. One of these, XP189MA, a 3-year-old girl with sun sensitivity, mental and physical developmental delay, has XPD mutations not previously reported, and barely detectable levels of nucleotide excision repair. The other, XP38BR, a 28-year-old woman with sun sensitivity, pigmentation changes and skin cancers typical of XP, has a mutation that has been identified previously, but only in TTD patients with no features of XP. The level of repair of UV damage in XP38BR is substantially higher than that in other patients with the same mutation. With both patients, polarized light microscopy revealed a 'tiger-tail' appearance of the hair, and amino acid analysis of the hair shafts show levels of sulfur-containing proteins intermediate between those of normal and TTD individuals. Our findings highlight the complexities of genotype-phenotype relationships in the XPD gene.

The major mechanism for the repair of DNA double-strand breaks (DSBs) in mammalian cells is non-homologous end-joining (NHEJ), a process that involves the DNA-dependent protein kinase [1] and [2], XRCC4 and DNA ligase IV [3], [3], [4] and [6]. Rodent cells and mice defective in these components are radiation-sensitive and defective in V(D)J-recombination, showing that NHEJ also functions to rejoin DSBs introduced during lymphocyte development [7] and [8]. 180BR is a radiosensitive cell line defective in DSB repair, which was derived from a leukaemia patient who was highly sensitive to radiotherapy [9], [10] and [11]. We have identified a mutation within a highly conserved motif encompassing the active site in DNA ligase IV from 180BR cells. The mutated protein is severely compromised in its ability to form a stable enzyme–adenylate complex, although residual activity can be detected at high ATP concentrations. Our results characterize the first patient with a defect in an NHEJ component and suggest that a significant defect in NHEJ that leads to pronounced radiosensitivity is compatible with normal human viability and does not cause any major immune dysfunction. The defect, however, may confer a predisposition to leukaemia.