Xeroderma pigmentosum complementation group A protein is driven to nucleotide excision repair sites by the electrostatic potential of distorted DNA

The presumed DNA-binding cleft of xeroderma pigmentosum group A (XPA) protein, a key regulatory subunit of the eukaryotic nucleotide excision repair complex, displays a distinctive array of 6 positively charged amino acid side chains. Here, the molecular function of these closely spaced electropositive residues has been tested by systematic site-directed mutagenesis. After the introduction of single amino acid substitutions, the mutants were probed for protein-DNA interactions in electrophoretic mobility shift and photochemical crosslinking assays. This analysis led to the identification of a critical hot-spot for DNA substrate recognition composed of two neighboring lysines at codons 141 and 179 of the human XPA sequence. The replacement of other basic side chains in the DNA interaction domain conferred more moderate defects of substrate binding. When the function of XPA was tested as a fusion product with either mCherry or green-fluorescent protein, a glutamate substitution of one of the positively charged residues at positions 141 and 179 was sufficient to decrease DNA repair activity in human fibroblasts. Thus, the removal of a single cationic side chain abolished DNA-binding activity and significant excision repair defects could be induced by single charge inversions on the XPA surface, indicating that this molecular sensor participates in substrate recognition by monitoring the electrostatic potential of distorted DNA repair sites.     

Effects of inhibitors on repair of DNA in normal human and xeroderma pigmentosum cells after exposure to x-rays and ultraviolet irradiation

Experiments were carried out to obtain direct evidence for the hypothesis that in human cells the repair of UV-damaged DNA is initiated by an incision step, and that this step is defective in cells from patients having Xeroderma pigmentosum (XP). The alkaline sucrose gradient centrifugation technique was used to detect breaks in the DNA.A decreased sedimentation velocity of the DNA was found after exposure of normal and XP cells to high doses of UV (5000 erg/mm²). Breaks were induced in the DNA by the UV irradiation without the action of an enzyme. After exposure of both types of cell to UV doses of 100–500 erg/mm², breaks that might occur by enzymic incision were not observed, possibly because of immediate rejoining.After single-strand breaks had been induced by X-rays, rejoining did not occur at temperatures lower than 22°. Rejoining was inhibited by KCN, 2,4-dinitrophenol, EDTA, iodoacetate and crystal violet. Actinomycin D, acriflavine and phleomycin, also tested as potential inhibitors of the repair process, induced breaks or conformational changes in the DNA of unirradiated normal and XP cells.Application to UV-exposed cells of conditions that inhibit the rejoining of breaks did not cause accumulation of breaks in the DNA. The results suggest a coordinated and sequential performance of the steps in the repair of each UV lesion by repair enzymes which may act as a complex.     

Nucleotide excision repair and cancer

Nucleotide excision repair (NER) is the most versatile and best studied DNA repair system in humans. NER can repair a variety of bulky DNA damages including UV-light induced DNA photoproducts. NER consists of a multistep process in which the DNA lesion is recognized and demarcated by DNA unwinding. Then, a ~28 bp DNA damage containing oligonucleotide is excised followed by gap filling using the undamaged DNA strand as a template. The consequences of defective NER are demonstrated by three rare autosomal-rezessive NER-defective syndromes: xeroderma pigmentosum (XP), Cockayne syndrome (CS), and trichothiodystrophy (TTD). XP patients show severe sun sensitivity, freckling in sun exposed skin, and develop skin cancers already during childhood. CS patients exhibit sun sensitivity, severe neurologic abnormalities, and cachectic dwarfism. Clinical symptoms of TTD patients include sun sensitivity, freckling in sun exposed skin areas, and brittle sulfur-deficient hair. In contrast to XP patients, CS and TTD patients are not skin cancer prone. Studying these syndromes can increase the knowledge of skin cancer development including cutaneous melanoma as well as basal and squamous cell carcinoma in general that may lead to new preventional and therapeutic anticancer strategies in the normal population.     

Semiautomated autoradiographic measurement of DNA repair in normal and xeroderma pigmentosum cultured human fibroblasts

Summary Assessment of DNA repair in cultured human fibroblasts by autoradiography may be facilitated by using semiautomated grain counting instruments. The instrument-determined number of autoradiographic grains per nucleus in cultured human skin fibroblasts was found to be linear in comparison to visual counts up to only 30 grains per nucleus. However, with two different instruments a greater range of linearity (to 100 to 120 grains per nucleus) was attained by measuring the grain surface area per nucleus. Semiautomated analysis of the grain surface area per nucleus yielded measurements of relative rates of unscheduled DNA synthesis after ultraviolet irradiation in xeroderma pigmentosum and normal human fibroblasts, which were reproducible and rapid.     

The inhibition of DNA repair by aphidicolin or cytosine arabinoside in X-irradiated normal and xeroderma pigmentosum fibroblasts

Normal and excision-deficient xeroderma pigmentosum fibroblasts were X-irradiated and the influence on DNA repair of either the repair inhibitor cytosine arabinoside or the specific inhibitor of Dna polymerase alpha, aphidicolin, investigated. The data indicated that the repair of a certain fraction of X-ray-induced lesions can be inhibited in both cell lines by both compounds. Thus, as aphidicolin blocks the operation of polymerase alpha, this enzyme must be involved in an excision repair pathway operating in both normal and excision-deficient xeroderma pigmentosum cells.     

Efficient repair of cyclobutane pyrimidine dimers at mutational hot spots is restored in complemented Xeroderma pigmentosum group C and trichothiodystrophy/xeroderma pigmentosum group D cells

Xeroderma pigmentosum (XP) and trichothiodystrophy (TTD) are rare heritable diseases. Patients suffering from XP and 50% of TTD afflicted individuals are photosensitive and have a high susceptibility to develop skin tumors. One solution to alleviating symptoms of these diseases is to express the deficient cDNAs in patient cells as a form of gene therapy. XPC and TTD/XPD cell lines were complemented using retroviral transfer. Expressed wild-type XPC or XPD cDNAs in these cells restored the survival to UVC radiation to wild-type levels in the respective complementation groups. Although complemented XP cell lines have been studied for years, data on cyclobutane pyrimidine dimer (CPD) repair in these cells at different levels are sparse. We demonstrate that CPD repair is faster in the complemented lines at the global, gene, strand specific, and nucleotide specific levels than in the original lines. In both XPC and TTD/XPD complemented lines, CPD repair on the non-transcribed strand is faster than that for the MRC5SV line. However, global repair in the complemented cell lines and MRC5SV is still slower than in normal human fibroblasts. Despite the slower global repair rate, in the complemented XPC and TTD/XPD cells, almost all of the CPDs at "hotspots" for mutation in the P53 tumor database are repaired as rapidly as in normal human fibroblasts. Such evaluation of repair at nucleotide resolution in complemented nucleotide excision repair deficient cells presents a crucial way to determine the efficient re-establishment of function needed for successful gene therapy, even when full repair capacity is not restored.     

Defective repair of mitomycin C crosslinks in Fanconi's anemia and loss in confluent normal human and xeroderma pigmentosum cells

Crosslink repair of mitomycin C-induced interstrand crosslinks was studied in exponentially growing and confluent normal human, transformed WI38CT-1, Fanconi's anemia (FA) and xeroderma pigmentosum (XP) group-A fibroblasts by the assay methods of alkaline sucrose centrifugation, hydroxyapatite column chromatography and S₁-nuclease digestion. These three methods demonstrated unequivocally that crosslinking occurred at a rate of 0.13 crosslinks/10⁸ Da per μg per ml mitomycin C ( 10 μg/ml) and the first half-excision of crosslinks followed the rapid first-order kinetics of 2–3 h half-life in exponentially-growing normal, WI38CT-1 and XP group-A cells. However, the first half-excision was completely defective in three out of the four FA strains tested and severely retarded in an FA strain. These results strongly support our previous observations in different strains of normal human, FA and XP group-A cells. An important new addition is that confluent, otherwise proficient, normal and XP cells almost completely lost the ability of the first, rapid half-excision of mitomycin C crosslinks in their DNA. This probably suggests that the enzyme or regulatory factor responsible for the half-excision, which differs from that for nucleotide excision repair, present constitutively in confluent cells, may be induced or activated only in the cycling cells. However, its relation to a defective FA factor is not clear at present.     

Comparative studies of host-cell reactivation, colony forming ability and excision repair after UV irradiation of xeroderma pigmentosum, normal human and some other mammalian cells

Host-cell reactivation, that is, the degree of survival of Herpes simplex virus after UV irradiation, was high in African green monkey BSC-1 cells, intermediate in normal human fibroblasts and human FL cells, and low in both xeroderma pigmentosum (XP) cells and mouse L cells. However, colony-forming ability after UV was high for FL, normal human fibroblasts and L cells, slightly low for BSC-1 cells and extremely low for XP cells. During the 24-h post-UV incubation period, up to about 50% of the thymine-containing dimers in the acid-insoluble DNA fraction disappeared at an almost equal rate for BSC-1, FL and normal human cells but remained unaltered for the XP cells. Alkaline sucrose gradient centrifugation of DNA after UV irradiation revealed only a slight difference between FL and BSC-1 cells in the kinetics of formation of single-strand breaks and their apparent repair. From these and the previously known characters of L cells possessing reduced excision-repair ability, if any, we may conclude that, if the survival of UV-irradiated Herpes simplex virus on a test line of human or other mammalian cells is as low as that on excisionless XP cells, then it is very probable that the test cell line is defective in excision repair. This reasoning leads to the presumptive conclusion that mouse L cells have an enhanced post-replication repair other than excision repair to deal with UV damage responsible for inactivation of colony-forming ability.     

High frequency of PTEN mutations in nevi and melanomas from xeroderma pigmentosum patients

We examined nevi and melanomas in 10 xeroderma pigmentosum (XP) patients with defective DNA repair. The lesions had a lentiginous appearance with markedly increased numbers of melanocytes. Using laser capture microdissection, we performed DNA sequencing of 18 benign and atypical nevi and 75 melanomas (melanoma in situ and invasive melanomas). The nevi had a similar high frequency of PTEN mutations as melanomas [61% (11/18) versus 53% (39/73)]. Both had a very high proportion of UV‐type mutations (occurring at adjacent pyrimidines) [91% (10/11) versus 92% (36/39)]. In contrast to melanomas in the general population, the frequency of BRAF mutations (11%, 7/61), NRAS mutations (21%, 13/62), and KIT mutations (21%, 6/28) in XP melanomas was lower than for PTEN. Phospho‐S6 immunostaining indicated activation of the mTOR pathway in the atypical nevi and melanomas. Thus, the clinical and histological appearances and the molecular pathology of these UV‐related XP nevi and melanomas were different from nevi and melanomas in the general population.     

Cell-type-specific level of DNA nucleotide excision repair in primary human mammary and ovarian epithelial cell cultures

DNA repair, a fundamental function of cellular metabolism, has long been presumed to be constitutive and equivalent in all cells. However, we have previously shown that normal levels of nucleotide excision repair (NER) can vary by 20-fold in a tissue-specific pattern. We have now successfully established primary cultures of normal ovarian tissue from seven women by using a novel culture system originally developed for breast epithelial cells. Epithelial cells in these cultures aggregated to form three-dimensional structures called "attached ovarian epispheres". The availability of these actively proliferating cell cultures allowed us to measure NER functionally and quantitatively by the unscheduled DNA synthesis (UDS) assay, a clinical test used to diagnose constitutive deficiencies in NER capacity. We determined that ovarian epithelial cells manifested an intermediate level of NER capacity in humans, viz., only 25% of that of foreskin fibroblasts, but still 2.5-fold higher than that of peripheral blood lymphocytes. This level of DNA repair capacity was indistinguishable from that of normal breast epithelial cells, suggesting that it might be characteristic of the epithelial cell type. Similar levels of NER activity were observed in cultures established from a disease-free known carrier of a BRCA1 truncation mutation, consistent with previous normal results shown in breast epithelium and blood lymphocytes. These results establish that at least three "normal" levels of such DNA repair occur in human tissues, and that NER capacity is epigenetically regulated during cell differentiation and development.     

UV-induced DNA repair synthesis in cells of patients with different forms of xeroderma pigmentosum and of heterozygotes

UV-induced DNA repair synthesis, as measured by autoradiography as well as by isopycnic centrifugation methods, was studied in a large number of cell strains from patients with the classic form of xeroderma pigmentosum (XP) or the De Sanctis-Cacchione syndrome (DSC) and several of their heterozygous parents. On the basis of the kinetics of repair synthesis in the cultured skin fibroblasts we have recognized four distinct groups of XP patients: (1) classic XP patients with low residual repair capacities, (2) classic XP patients with intermediate, but dose-dependent, levels of repair synthesis relative to the normal level, (3) patients, diagnosed as having classic XP, with a normal or only slightly reduced repair capacity and (4) DSC patients with a complete deficiency of repair synthesis. Complementation studies reported elsewhere have shown that different mutations are responsible for the detect in at least three of these groups. Cell strains of each of the four XP types were able to rejoin single-strand DNA breaks induced by X-rays. Most of the cell strains derived from heterozygotes showed normal repair activities. However, in the parents some of the of DSC-patients a significant reduction of the level of repair synthesis was found.     

A rapid procedure for measurement of DNA repair in human fibroblasts and for complementation analysis of xeroderma pigmentosum cells

A rapid procedure for measuring unscheduled DNA synthesis has been studied in detail. Human fibroblasts were brought into the non-dividing state by either growing to confluence or starvation for arginine. Residual semi-conservative synthesis was abolished by hydroxyurea. Hydroxyurea-resistant DNA synthesis which was induced by irradiation and chemical mutagens was presumed to represent repair synthesis and provided a very rapid semi-quantitative procedure for its measurement. Problems were encountered, however, when comparing the quantitative response of different cell strains. The variability between experiments was quite large, and we found that the level of repair synthesis depended not only on the mutagen and the genotype of the cell, but also on physiological factors. This led to some anomalous results. The system was able to detect with ease the large defects in UV-induced repair synthesis in fibroblasts from patients with xeroderma pigmentosum (XP) but it would probably not easily detect less than a 50% reduction in the level of repair synthesis. By extension of this procedure, in combination with cell fusion induced by polyethylene glycol, we have developed a method for carrying out genetic complementation of XP fibroblasts, which does not entail the use of either Sendai virus or of autoradiography. Results of complementation analysis of 4 XP cell strains are presented.     

Alterations in levels of 5'-adenyl dinucleotides following DNA damage in normal human fibroblasts and fibroblasts derived from patients with xeroderma pigmentosum

Levels of 5'-adenyl dinucleotides, measured as diadenosine-5',5'-P1,P4-tetraphosphate (Ap4A), were found to accumulate in cultured human fibroblasts following treatment with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), the radiomimetic drug bleomycin, and nitroquinoline-1-oxide (NQO) or UV-irradiation in the presence of cytosine arabinofuranoside (araC). In contrast, cells derived from patients with xeroderma pigmentosum complementation group A (XP-A) did not demonstrate an increase in DNA-strand breaks following UV irradiation or NQO in the presence of araC nor an increase in Ap4A levels. Ap4A accumulation did occur in XP-A cells following treatment with MNNG. Cells derived from patients characterized as XP variants, which are incision repair-proficient, accumulated 5'-dinucleotides following bleomycin, MNNG and UV or NQO in the presence of araC. Taken together, these data suggest that Ap4A accumulates as a response to DNA-strand breaks.     

Single-strand breaks in DNA during repair of UV-induced damage in normal human and xeroderma pigmentosum cells as determined by alkaline DNA unwinding and hydroxylapatite chromatography: effects of hydroxyurea, 5-fluorodeoxyuridine and 1-beta-D-arabinofuranosylcytosine on the kinetics of repair.

A simple and sensitive technique for detection of strand breaks in DNA has been further developed. The method has been used to follow UV-induced excision-repair in human fibroblasts. It has been possible to study the kinetics of enzymic reactions in intact cells, in which strand breaks in DNA are produced and sealed again. Hydroxyurea, 5-fluorodeoxyuridine and 1-beta-D-arabinofuranosylcytosine, potent inhibitors of DNA synthesis, drastically increased the number of breaks observed during the repair process. This was probably due to a decreased polymerase activity, which will cause the strand breaks formed by endonuclease to remain open longer. The initial rate of strand-break formation did not seem to be influenced by hydroxyurea or araC, and was about 4000 breaks per minute in a diploid genome, at a dose of 20 J/m2. After 5--30 min, depending on the dose of UV, the number of breaks reached a maximum and started to decrease again. Hydroxyurea decreased the rate of polymerization in the sites under repair. However, there was no concomitant reduction of repair-induced incorporation of [3H]thymidine and no reduction of the excision of pyrimidine dimers. It therefore seems that the action of the polymerase was not a rate-limiting event, but rather an earlier step. It is likely that the endonucleolytic activity determined the rate of repair. As a consequence, the endonuclease and polymerase cannot be bound in a permanent complex. Under certain assumptions, the time for repair of a site, i.e. the time from incision to final ligase sealing, can be estimated as between 3 and 10 min. Essentially no breaks were produced in Xeroderma pigmentosum cells belonging to complementation group A, and there was no enhancement by hydroxyurea. Cells from the variant type of Xeroderma pigmentosum behaved like normal cells in this respect.     

Induction of unscheduled DNA synthesis by the carcinogen 7-bromomethylbenz(a)anthracene and its removal from the DNA of normal and xeroderma pigmentosum lymphocytes

The carcinogen 7-bromomethylbenz(a)anthracene (BBA), which can bind strongly to DNA, induces unscheduled DNA synthesis (DNA repair) in normal lymphocytes but almost none in lymphocytes from patients with Xeroderma pigmentosum (XP), and inherited disease known to be defective in excision repair of ultraviolet-damaged DNA. We studied [³H]BBA's ability to bind to DNA of normal and XP lymphocytes, its influence on unscheduled DNA synthesis, and its removal from the DNA of both cell types. We found that 20–30% of the BBA is bound to macromolecules other than DNA and that its binding to DNA is essentially complete after 30 min. The induction of unscheduled DNA synthesis by the carcinogen in XP lymphocytes was approximately 10% of that induced in normal lymphocytes. While 15–20% of the BBA was removed from the DNA of normal cells 6 h after treatment, only 1–2% was removed from the DNA of XP cells. Thus, XP cells not only are defective in repairing ultraviolet-damaged DNA and excising thymine dimers but also fail to repair DNA damaged by certain carcinogens, and, most importantly, fail to remove the DNA-bound carcinogen, BBA.     

Systems biology approach for mutational and site-specific structural investigation of DNA repair genes for xeroderma pigmentosum

Xeroderma pigmentosum (XP) is a rare genetic skin disorder caused due to the extreme sensitivity for ultraviolet (UV) radiations. On its exposure, DNA acquires damages leading to skin and often neurological abnormalities. The DNA repair implicated in fixing UV-induced damages is NER and mutations in genes involved in NER and TLS form the basis of XP. The analyses of such mutations are vital for understanding XP and involved cancer genetics to facilitate the identification of crucial biomarkers and anticancer therapeutics. We detected the deleterious nsSNPs and examined them at structure-level by altering the structure, estimating secondary structure, solvent accessibility and performing site specific analysis. Crucial phosphorylation sites were also identified for their role in the disorder. These mutational and structural analyses offer valuable insight to the fundamental association of genetic mutations with phenotypic variations in XP and will assist experimental biologists to evaluate the mutations and their impact on genome.     

Genetic polymorphisms of xeroderma pigmentosum group D gene Asp312Asn and Lys751Gln and susceptibility to prostate cancer: A systematic review and meta-analysis

Many studies have reported the role of xeroderma pigmentosum group D (XPD) with prostate cancer risk, but the results remained controversial. To derive a more precise estimation of the relationship, a meta-analysis was performed. Odds ratios (ORs) with 95% confidence intervals (CIs) were estimated to assess the association between XPD Asp312Asn and Lys751Gln polymorphisms and prostate cancer risk. A total of 8 studies including 2620 cases and 3225 controls described Asp312Asn genotypes, among which 10 articles involving 3230 cases and 3582 controls described Lys751Gln genotypes and were also involved in this meta-analysis. When all the eligible studies were pooled into this meta-analysis, a significant association between prostate cancer risk and XPD Asp312Asn polymorphism was found. For Asp312Asn polymorphism, in the stratified analysis by ethnicity and source of controls, prostate cancer risk was observed in co-dominant, dominant and recessive models, while no evidence of any associations of XPD Lys751Gln polymorphism with prostate cancer was found in the overall or subgroup analyses. Our meta-analysis supports that the XPD Asp312Asn polymorphism contributed to the risk of prostate cancer from currently available evidence. However, a study with a larger sample size is needed to further evaluate gene–environment interaction on XPD Asp312Asn and Lys751Gln polymorphisms and prostate cancer risk.