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Chapter 25 : Other Diseases Associated with Defects in Nucleotide Excision Repair of DNA
Category: Microbial Genetics and Molecular Biology
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This chapter discusses diseases associated with defects in nucleotide excision repair (NER) of DNA. It first talks about clinical phenotypes and cellular phenotypes of cockayne syndrome (CS). Genes designated CSA and CSB (representing the two complementation groups) have been isolated and characterized, and mutations have been identified in either of these two genes in all CS cases examined. Other clinical entities associated with mutations in CS or xeroderma pigmentosum (XP) genes are considered. The clinical entities discussed are cerebro-oculo-facio-skeletal (COFS) syndrome, UV-sensitive syndrome, and transcription syndromes. The biochemical and clinical consequences of different mutations in a single gene are often unpredictable and can produce different clinical phenotypes. Such is the case with both the combined XP/CS complex and another related disease called trichothiodystrophy (TTD) (or PIBIDS). The general considerations with respect to understanding the combined XP/CS complex apply to the relationship between XP and TTD.
Cells from patients with CS are hypersensitive to killing by UV radiation.
Cells from individuals with CS (fibroblasts are shown here) are slightly more sensitive than normal cells to killing following exposure to ionizing radiation. (Adapted from reference 156 .)
CS cells in culture manifest reduced ability to express a reporter gene (chloramphenicol acetyltransferase [CAT]) in a plasmid construct after exposure of the plasmid to UV radiation. WT, wild type. (Adapted from reference 131 .)
CS cells are hypermutable (shown here as mutations scored as thioguanine resistance [TGr]) following exposure to UV radiation. XP cells are shown for comparison. (Adapted from reference 5 .)
When cells from individuals with CS are exposed to UV radiation, the recovery of total RNA synthesis (measured by the amount of [3H]uridine incorporated into cells) is delayed relative to that observed in normal cells.
CS cells (B) are defective in preferential NER of photoproducts from DNA compared to normal cells (A). TS, transcribed strand; NTS, nontranscribed strand. (Adapted from reference 163 .)
CS cells manifest increased apoptosis following exposure to UV radiation. This phenotype is substantially corrected following transfection with the appropriate wild-type gene. (Adapted from reference 135 .)
Genetic complementation groups for CS were determined by demonstrating correction of reduced rates of RNA synthesis after fusion of different CS cell lines. Correction of the phenotype in any fusion experiment implies that the two cell lines carry defects in different CS genes. RNA synthesis was measured by quantitating grain counts in autoradiograms. Monokaryons (monos) are shown as gold bars; bikaryons are shown as dark grey bars; multikaryons (multis) are shown as light grey bars. The CS numbers refer to particular cell lines from CS individuals. (Adapted from reference 83 .)
Correction of the phenotypes of UV radiation sensitivity (A) and recovery of defective RNA synthesis (B) in CS-B cells transfected with the cloned CSB (ERCC6) gene. Solid black line, wild-type cells; dotted black line, CS-A cells; dotted gold lines, complemented CS-A cells. (Adapted from reference 154 .)
Correction of the phenotype of sensitivity to UV radiation sensitivity of CS-A cells by the cloned CSA gene, measured by expression of chloramphenicol acetyltransferase (CAT) activity from a UV-irradiated reporter plasmid. Solid grey line, wild-type cells; solid black line, CS-A cells; dotted black line, complemented CS-A cells. (Adapted from reference 67 .)
UV radiation-induced ubiquitination of the large subunit of RNA polymerase II in human cells in normal, CS-A, and CS-B cells. Pol IIo, hypophosphorylated form of RNA polymerase II; Pol IIa, hyperphosphorylated form of RNA polymerase II; x, ubiquitinated species of RNA polymerase II. Both CS-A and CS-B cells were transfected with an empty vector (control construct) or one carrying the CSA or CSB cDNA. (Adapted from reference 17 .)
UV radiation-induced translocation of CSA protein to the nucleus in CS-A cells expressing the CSA gene. Cells were exposed to UV radiation and incubated for various periods. Cell extracts were subjected to specific treatments (lane numbers) to isolate various cytoplasmic and nuclear fractions, and these were then examined by gel electrophoresis. CSA protein was detected by Western blotting. Lane 9 represents a nuclear matrix fraction. (Adapted from reference 74 .)
Defective transcription-coupled NER of the Trp53 gene in Csb-defective mouse cells. +/ + , wild-type mice; +/ —, heterozygous mutant mice; —/ —, homozygous mutant mice. (Adapted from reference 159 .)
Kinetics of skin tumor formation in Csb +/+ (dark gold line), Csb+/— (grey line), and Csb—/— (light gold line) mice following exposure of the shaved skin to UV radiation. (Adapted from reference 159 .)
Predisposition of wild-type, Csa mutant, Xpc mutant, and Csa Xpc double-mutant mice to skin cancer following exposure of the shaved dorsal skin to UV radiation. (Adapted from reference 158 .)
Csa Xpa double-mutant mice (left) are runted and develop very slowly compared to wild-type control animals (right). (Courtesy of G. T. J. van der Horst.)
Complementation of UV radiation sensitivity in cells from a UVS syndrome individual. WT, wild type. (Adapted from reference 67a .)
TTD patients manifest cleavage fractures of the hair shafts (trichoschisis). Also note the slightly undulating contour of the hair shaft and the wavy distribution pattern of melanin granules in the hair cortex. The latter feature reflects the varying orientation of the internal fiber structure in TTD.
Alternating light and dark (tiger tail) pattern of the hair shaft as observed under polarizing light, from a patient with TTD. (Adapted from reference 164 with permission.)
Quantitative expression of repair synthesis levels in UV-irradiated TTD homozygous (TTD) and heterozygous (TTDH) G0 lymphocytes.
(Left) Photograph of a TTD patient 10 days after a febrile episode associated with pneumonia. (Right) The same patient 10 weeks later. (Adapted from reference 165 with permission.)
Figure 25-22 A TTD patient with β-thalassemia shows significantly different ratios of β/α-globin mRNA in cells compared to normal (control) cells and cells from a patient with XP. (Adapted from reference 167 with permission.)
Mouse with TTD. Note the hair loss and reduced body size compared to a normal animal. (Courtesy of J. H. J. Hoeijmakers and reproduced with permission.)
Effect of anti-XAB2 antiserum microinjection on DNA repair (UDS and RRS) and transcription a
Complementation analysis of heterokaryons obtained by fusion of TTD and other human fibroblasts