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Chapter 24 : Xeroderma Pigmentosum
Category: Microbial Genetics and Molecular Biology
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Understanding how DNA repair operates in human cells was for many years limited by the availability of mutant cell lines genetically defective in various responses to DNA damage. A researcher in University of California at San Francisco obtained skin biopsy specimens from xeroderma pigmentosum (XP) individuals and discovered that cultures of fibroblasts derived from such patients are defective in repair synthesis following exposure to UV radiation, suggesting that they are indeed defective in nucleotide excision repair (NER). These observations were independently confirmed by another research group and provided the first demonstration of a DNA repair defect associated with a hereditary human disease. The group's observations of XP cells provided an impetus to examine the response to DNA-damaging agents in cells derived from other hereditary human diseases, particularly those associated with spontaneous or environmentally induced chromosomal abnormalities or with an abnormally high incidence of neoplasia. In addition, the observation that human subjects suffering from XP in particular are severely prone to skin tumors associated with sunlight exposure prompted further exploration of the relationship between DNA damage, mutagenesis, and neoplastic transformation in a more general sense. Since then, an enormous amount of information has been garnered about the molecular pathology of XP. This chapter considers mutant mouse strains defective in gene functions required for NER, some (but not all) of which mimic some of the phenotypes of humans with XP. XP mouse strains defective in the Xpa, Xpc, Xpd, Xpe, Xpg, Rad23A, Rad23B, and Ercc1 genes have been generated.
Defective repair synthesis (UDS) of DNA can be demonstrated in the epidermal cells of living XP individuals following the injection of tritiated thymidine into an area of skin previously exposed to UV radiation. The panel on the left is from an unirradiated normal subject, and that in the middle from a UV-irradiated normal subject. The panel on the right is from a UV-irradiated XP individual.
In some cases of severe Sun exposure, cancers of the tongue can develop in individuals with XP.
(A) Age at which first skin cancer was reported for 186 XP individuals compared with the age distribution for over 29,000 non-XP patients with either basal or squamous cell carcinoma in the U.S. general population. (B) Age distribution of patients with XP. The age at last clinical observation is shown for 785 patients, 373 of whom were also reported to have skin cancer.
XP cells in culture from most genetic complementation groups are hypersensitive to UV radiation. However, the precise level of sensitivity varies somewhat from cell line to cell line within a given genetic complementation group and particularly between genetic complementation groups.
XP cells (genetic complementation groups A and C are shown here) have an increased frequency of mutations at various genetic loci (such as the locus for azaguanine resistance shown) when exposed to DNA-damaging agents such as UV radiation.
The kinetics of the disappearance of sites in DNA (pyrimidine dimers) that are sensitive to attack by a pyrimidine dimer-DNA glycosylase (see chapter 7 for details of this technique). The percentages shown are relative to the enzyme-sensitive sites (ESS) detected in unirradiated cells. The broken line in each panel is reproduced from the kinetics observed with normal cells in the first panel.
Defective repair synthesis (UDS) in XP cells in culture. Normal cells (left panel) show autoradiographic labeling of the great majority of the nuclei that are not in S phase (intensely labeled cells), reflecting repair synthesis of DNA. XP cells (right panel) show normal S-phase (scheduled) DNA synthesis, but are defective in UDS. (Courtesy of J. E. Cleaver.)
Host cell reactivation of UV-irradiated plasmid DNA measured by chloramphenicol acetyltransferase (CAT) activity in a reporter plasmid following transfection of XP cells with wild-type XP genes. (Left) XP-A cells transfected with the XPA (light gold) or XPC (dark gold) gene. (Right) XP-F cells transfected with the XPF (light gold) or XPC (dark gold) gene. (Adapted from reference 167 .)
DNA containing oxidative base damage produced by exposure to γ-rays (A) or H2O2 plus Cu2+ (B) is repaired by extracts of normal cells but not by extracts of cells from XP complementation group A.
Repair of UV-B radiation-induced DNA damage in lymphocytes from various XP genetic complementation groups. Repair is shown as relative chloramphenicol acetyltransferase (CAT) activity from a reporter gene transfected into the cells. Black lines show the means of multiple independent experiments in a given cell line, and gold lines reflect mean values with eight different cell lines from a given XP complementation group. The shaded areas indicate the minimum and maximum values of the wild-type range. (Adapted from reference 203 .)
Catalase activity measured in extracts of cells from normal, XP heterozygous, and XP homozygous individuals of different genetic complementation groups. (Adapted from reference 253 .)
Correlation between mean catalase activity in cell extracts and DNA repair efficiency, expressed as relative UDS after exposure to UV light. Cells were transfected with viruses carrying the indicated XP genes. het, heterozygous. (Adapted from reference 253 .)
Structure of the R and S diastereoisomers of 5’,8-cyclo-2’-deoxyadenosine. These structures are found in DNA damaged by hydroxyl radicals. These lesions contain two covalent linkages to the sugar-phosphate backbone of DNA. (Adapted from reference 138 .)
Plasmid DNA containing 5’,8-cyclo-2’-deoxy-adenosine residues in either the R (gold bars) or S (grey bars) form was incubated with extracts of cells proficient for NER in the absence or presence of XPA antiserum (to inactivate NER). The figure shows quantitation of the amount of oligonucleotide excision product generated by NER. ab., antibody; prot., protein. (Adapted from reference 138 .)
Complementation of defective repair synthesis (UDS) in heterodikaryons derived by fusing cells of XP individuals from different genetic complementation groups. The cells labeled a to d are monokaryons which are defective in UDS. The cells labeled f and g are homodikaryons resulting from fusion of cells from the same individual and hence are also defective in UDS. The heterodikaryon labeled e shows restoration of normal levels of UDS in both nuclei. (Courtesy of J. E. Cleaver.)
Quantitative complementation analysis by monitoring UDS after treatment of cells with either UV radiation or the UV radiomimetic chemical 4-nitroquinoline 1-oxide (4-NQO). When cells from two group C individuals are fused (C/C fusion), the levels of UDS in the heterodikaryons are no greater than in each of the monokaryons. However, when XP-A and XP-D cells are fused (A/D fusion), the levels of UDS in the heterodikaryons are greater than in each of the monokaryons.
Autoradiograph showing that expression of the XPB gene following its microinjection into the nuclei of TTD6V1 cells results in the correction of defective UDS in these cells. Two uncomplemented cells can also be seen to the right. (Courtesy of W. Vermeulen and Jan H. J. Hoeijmakers.)
Distribution of different base substitutions observed in internal and skin tumors from non-XP individuals and in skin tumors from XP-C individuals. (Adapted from reference 86 .)
Diagrammatic representation of the relative locations on mutations identified in a group of 17 XP individuals from genetic complementation group D, resulting either in classic XP or TTD. Amino acid changes in individual cell lines are boxed in grey, and white boxes show cell line designations in which the subscripts 1 and 2 indicate different alleles. (A) Mutations found in all the cell lines, with XP and TTD lines segregated above and below the stick-like polypeptide, respectively. The small gold bars beneath the stick-like polypeptide indicate the relative sizes of deletion mutations. (B) Subset of mutations found in both XP-D and TTD individuals. I to VII denote the seven DNA helicase domains in the primary structure of the XPD protein. (Adapted from reference 239 .)
Mutations in the DDB2 (XPE) gene in eight XP-E individuals. The p48 ORF is diagrammatically represented with exons indicated in roman numerals. Putative WD domains in the protein are shown as light gold boxes, and the dark gold boxes represent putative nuclear localization signals. Mutations in DNA are shown above or below boxes, which contain corresponding amino acid (aa) changes. All the mutations map to the C-terminal half of the protein. (Adapted from reference 195 .)
Rates of new actinic keratoses in placebo-treated and T4 endonuclease V-treated individuals. (Adapted from reference 267 .)
Kinetics of tumor development in Xpa mice defective in NER following exposure to UV-B radiation (left) or 7,12-dimethylbenz[a]anthracene (DMBA) (right). The black lines represent wild-type mice, and the grey line represents Xpa+/- mice. The gold line represents Xpa-/- mice. (Adapted from reference 58 .)
Mutagenesis in lymphocytes from wild-type (gold line) and Xpa -/- mutants (black line) following exposure to benzo[a]pyrene [B(a)P]. (Adapted from reference 12 .)
Cells from Xpc-/- mice are defective in NER of base damage [(6–4)PP] in the nontranscribed DNA strand (NTS) of the Trp53 (p53) gene following exposure to UV radiation. TS, transcribed strand. (Adapted from reference 29 .)
UV-B radiation-induced skin cancer in Xpc-/- mice. Animals were exposed to daily doses of UV-B radiation for several weeks. Skin cancers develop on the shaved dorsal skin in Xpc-/- mice much more rapidly than in heterozygous mutant or wild-type mice.
Xpc-/-mice (dark gold line) develop skin cancer much faster than Xpc +/- (black line) or wild-type (light gold line) animals do. However, after about 40 weeks, Xpc +/- animals show an increased skin cancer predisposition relative to wild-type mice.
The loss of one Trp53 (p53) allele sensitizes both Xpc +/- and Xpc -/- mutant mice to UV-B radiation-induced skin cancer.
Spectrum of some of the more prominent mutations in the nontranscribed strand of the Trp53 (p53) gene of skin tumors from UV-B-irradiated mice with the indicated genotypes. Notice that the threonine codon 122 (T122), located at the end of codon 4, is a very hot spot for mutations uniquely in Xpc -/- Trp53+ /- animals. Mutations at codons 124 and 210 observed in various Xpc mutant mice have not been previously found in skin tumors in mice.
Frequency of mutations in codon 122 of the Trp53 gene in wild-type and Xpc mutant mice of various genotypes, relative to mutations at all other locations in the Trp53 gene. Mutations were determined in the Trp53 gene from UVB radiation-induced skin cancers.
Xpc homozygous mutant mice are highly prone to liver and lung tumors after exposure to acetylaminofluorene. (A) The liver from an Xpc -/- mutant is riddled with tumors. (B) Incidence of both liver and lung tumors in mice of various genotypes.
Frequency of spontaneous mutations at the Hprt locus in lymphocytes in mice of the indicated genotypes. Mutations were measured at 12 months of age. (Adapted from reference 260 .)
Phenotypic rescue of viability following expression of an Ercc1 transgene in the liver of Ercc1 -/- mice. (Adapted from reference 220 .)