Proteolysis of a nucleotide excision repair protein by the 26 S proteasome

The 26 S proteasome degrades a broad spectrum of proteins and interacts with several nucleotide excision repair (NER) proteins, including the complex of Rad4 and Rad23 that binds preferentially to UV-damaged DNA. The rate of NER is increased in yeast strains with mutations in genes encoding subunits of the 26 S proteasome, indicating that it could negatively regulate a repair process. The specific function of the 26 S proteasome in DNA repair is unclear. It might degrade DNA repair proteins after repair is completed or act as a molecular chaperone to promote the assembly or disassembly of the repair complex. In this study, we show that Rad4 is ubiquitylated and that Rad23 can control this process. We also find that ubiquitylated Rad4 is degraded by the 26 S proteasome. However, the interaction of Rad23 with Rad4 is not only to control degradation of Rad4, but also to assist in assembling the NER incision complex at UV-induced cyclobutane pyrimidine dimers. We speculate that, following the completion of DNA repair, specific repair proteins might be degraded by the proteasome to regulate repair.     

Heterogeneity and overlaps in nucleotide excision repair disorders

Nucleotide excision repair (NER) is an essential DNA repair pathway devoted to the removal of bulky lesions such as photoproducts induced by the ultraviolet (UV) component of solar radiation. Deficiencies in NER typically result in a group of heterogeneous distinct disorders ranging from the mild UV sensitive syndrome to the cancer-prone xeroderma pigmentosum and the neurodevelopmental/progeroid conditions trichothiodystrophy, Cockayne syndrome and cerebro-oculo-facio-skeletal-syndrome. A complicated genetic scenario underlines these disorders with the same gene linked to different clinical entities as well as different genes associated with the same disease. Overlap syndromes with combined hallmark features of different NER disorders can occur and sporadic presentations showing extra features of the hematological disorder Fanconi Anemia or neurological manifestations mimicking Hungtinton disease-like syndromes have been described. Here, we discuss the multiple functions of the five major pleiotropic NER genes (ERCC3/XPB, ERCC2/XPD, ERCC5/XPG, ERCC1 and ERCC4/XPF) and their relevance in phenotypic complexity. We provide an update of mutational spectra and examine genotype-phenotype relationships. Finally, the molecular defects that could explain the puzzling overlap syndromes are discussed.     

The nucleotide excision repair epistasis group in Neurospora crassa

Summary DNA repair mutants in eucaryotes are normally assigned to three epistasis groups. Each epistasis group represents a pathway for DNA repair. The pathways are commonly designated (1) nucleotide excision repair, (2) recombination repair and (3) mutagenic repair. An excision repair epistasis group has been established in Neurospora and the mutants assigned to this group should be limited in their ability to excise pyrimidine dimers and other bulky lesions from DNA. Using a pyrimidine dimer-specific assay, we have found that all Neurospora crassa mutants assigned to the excision repair epistasis group are capable of removing pyrimidine dimers from the DNA at a rate similar to the wild-type organism.     

A multistep damage recognition mechanism for global genomic nucleotide excision repair

A mammalian nucleotide excision repair (NER) factor, the XPC-HR23B complex, can specifically bind to certain DNA lesions and initiate the cell-free repair reaction. Here we describe a detailed analysis of its binding specificity using various DNA substrates, each containing a single defined lesion. A highly sensitive gel mobility shift assay revealed that XPC-HR23B specifically binds a small bubble structure with or without damaged bases, whereas dual incision takes place only when damage is present in the bubble. This is evidence that damage recognition for NER is accomplished through at least two steps; XPC-HR23B first binds to a site that has a DNA helix distortion, and then the presence of injured bases is verified prior to dual incision. Cyclobutane pyrimidine dimers (CPDs) were hardly recognized by XPC-HR23B, suggesting that additional factors may be required for CPD recognition. Although the presence of mismatched bases opposite a CPD potentiated XPC-HR23B binding, probably due to enhancement of the helix distortion, cell-free excision of such compound lesions was much more efficient than expected from the observed affinity for XPC-HR23B. This also suggests that additional factors and steps are required for the recognition of some types of lesions. A multistep mechanism of this sort may provide a molecular basis for ensuring the high level of damage discrimination that is required for global genomic NER.     

p53-dependent global nucleotide excision repair of cisplatin-induced intrastrand cross links in human cells

Cisplatin is an extremely effective chemotherapeutic agent used for the treatment of testicular and other solid tumours. It induces a variety of structural modifications in DNA, the most abundant being the GpG- and ApG-1,2-intrastrand cross links formed between adjacent purine bases. These cross links account for approximately 90% of cisplatin-induced DNA damage and are thought to be responsible for the cytotoxic activity of the drug. In human cells, the nucleotide excision repair (NER) process removes the intrastrand cross links from the genome, the efficiency of which is likely to be an important determinant of cisplatin cytotoxicity. We have investigated whether the p53 tumour suppressor status affects global NER of cisplatin-induced intrastrand cross links in human cells. We have used a (32)P-postlabelling method to monitor the removal of GpG- and ApG-intrastrand cross links from two human cell models (the 041TR system, in which p53 is regulated by a tetracycline-inducible promoter, together with WI38 fibroblasts and the SV40-transformed derivative VA13) that each differ in p53 status. We demonstrate that the absence of functional p53 leads to persistence of both cisplatin-induced intrastrand cross links in the genome, suggesting that p53 regulates NER of these DNA lesions. This observation extends the role of p53 in NER beyond enhancing the removal of environmentally induced DNA lesions to include those of clinical origin. Given the frequency of p53 mutations in human tumours, these results may have implications for the use of cisplatin in cancer chemotherapy.     

Role for nucleotide excision repair in virulence of Mycobacterium tuberculosis

Mutations in Mycobacterium tuberculosis uvrB result in severe sensitivity to acidified nitrite, a source of nitric oxide (6). In this study, we show that a uvrB mutant is exquisitely sensitive to UV light but not to several sources of reactive oxygen species in vitro. Furthermore, a uvrB mutant was attenuated in mice as judged by an extension of life span. Attenuation in mice was partially reversed by genetic inactivation of nitric oxide synthase 2 (iNOS) and almost completely reversed in mice lacking both iNOS and phagocyte oxidase. Thus, a gene predicted to encode a key element of DNA repair is required for resistance of M. tuberculosis to both reactive nitrogen and reactive oxygen species in mice.     

The evolution and mechanisms of nucleotide excision repair proteins

Nucleotide excision repair (NER) pathways remove a wide variety of bulky and helix-distorting lesions from DNA, and involve the coordinated action of damage detection, helicase and nuclease proteins. Most archaeal genomes encode eucaryal-type NER proteins, including the helicases XPB and XPD and nuclease XPF. These have been a valuable resource, yielding important mechanistic and structural insights relevant to human health. However, the nature of archaeal NER remains very uncertain. Here we review recent studies of archaeal NER proteins relevant to both eucaryal and archaeal NER systems and the evolution of repair pathways.     

Inhibiting replication of begomoviruses using artificial zinc finger nucleases that target viral-conserved nucleotide motif

Geminiviridae consists of a large group of single-stranded DNA viruses that cause tremendous losses worldwide. Frequent mixed infection and high rates of recombination and mutation allow them to adapt rapidly to new hosts and overcome hosts’ resistances. Therefore, an effective strategy able to confer plants with resistance against multiple begomoviruses is needed. In the present study, artificial zinc finger proteins were designed based on a conserved sequence motif of begomoviruses. DNA-binding affinities and specificities of these artificial zinc fingers were evaluated using electrophoretic mobility shift assay. Artificial zinc finger nuclease (AZFNs) were then constructed based on the ones with the highest DNA-binding affinities. In vitro digest and transient expression assay showed that these AZFNs can efficiently cleave the target sequence and inhibit the replication of different begomoviruses. These results suggest that artificial zinc finger protein technology may be used to achieve resistance against multiple begomoviruses.     

DNA damage and nucleotide excision repair capacity in healthy individuals

Interindividual differences in DNA repair capacity (DRC) represent an important source of variability in genome integrity and thus influence health risk. In the last decade, DRC measurement has attracted attention as a potential biomarker in cancer prediction. Aim of the present exploratory study was to characterize the variability in DNA damage and DRC on 100 healthy individuals and to identify biological, lifestyle, or genetic factors modulating these parameters. The ultimate goal was to obtain reference data from cancer‐free population, which may constitute background for further investigations on cancer patients. The endogenous DNA damage was measured as a level of DNA single‐strand breaks and DRC, specific for nucleotide excision repair (NER), was evaluated using modified comet assay, following the challenge of peripheral blood mononuclear cells with benzo[a]pyrene diolepoxide. Additionally, genetic polymorphisms in NER genes (XPA, XPC, XPD, and XPG) were assessed. We have observed a substantial interindividual variability for both examined parameters. DNA damage was significantly affected by gender and alcohol consumption (P = 0.003 and P = 0.012, respectively), whereas DRC was associated with family history of cancer (P = 0.012). The stratification according to common variants in NER genes showed that DNA damage was significantly modulated by the presence of the variant T allele of XPC Ala499Val polymorphism (P = 0.01), while DRC was modulated by the presence of the A allele of XPA G23A polymorphism (P = 0.048). Our results indicate the range of endogenous DNA single‐strand breaks and capacity of NER in healthy volunteers as well as the role of potentially relevant confounders. Environ. Mol. Mutagen. 2011. © 2011 Wiley‐Liss, Inc.     

Differentiation-dependent p53 regulation of nucleotide excision repair in keratinocytes.

The role of the tumor suppressor p53 in repair of ultraviolet light (UV)-induced DNA damage was evaluated using a host-cell reactivation (HCR) assay. HCR determines a cell's ability to repair UV-damaged DNA through reactivation of a transfected CAT reported plasmid. Most UV damage is removed through nucleotide excision repair (NER). Primary murine keratinocytes isolated from p53-deficient and wild-type p53 mice were used in the HCR assay. The NER was reduced in p53-/- keratinocytes as compared with p53+/+ keratinocytes. The reduced DNA repair in p53-/- mice was confirmed with a radioimmunoassay comparing cyclobutane dimers (CPDs) and (6-4) photoproducts in p53+/+ and p53-/- keratinocytes after the cells were exposed to UV irradiation. Our results demonstrate that wildtype p53 plays a significant role in regulating NER. Furthermore, as there is evidence that p53 protein levels decrease after keratinocytes become differentiated, we sought to determine whether p53 plays a role in NER in differentiated keratinocytes. Differentiation of the keratinocytes by increasing the Ca2+ concentration in the culture media resulted in a marked reduction in NER equally in both p53+/+ and p53-/- groups. This finding suggests that reduced DNA repair after differentiation is p53 independent. A similar reduction in HCR was confirmed in differentiated human keratinocytes. These data, taken together, indicate that p53 or p53-regulated proteins enhance NER in basal undifferentiated keratinocytes but not in differentiated cells. As nonmelanoma skin cancers originate from the basal keratinocytes, our findings suggest that loss of p53 may contribute to the pathogenesis of this common skin cancer.     

Effects of nitrous acid treatment on the survival and mutagenesis of Escherichia coli cells lacking base excision repair(hypoxanthine-DNA glycosylase-ALK A protein) and/or nucleotide excision repair

Deoxyinosine occurs in DNA by spontaneous deamination of adenineor by incorporation of dITP during replication. Hypoxanthineresidues (HX) are mutagenic and give rise to A-T     

Alkaline unwinding flow cytometry assay to measure nucleotide excision repair

Nucleotide excision repair (NER), one of the DNA repair pathways, is the primary mechanism for repair of bulky adducts caused by physical and chemical agents, such as UV radiation, cisplatin and 4-nitroquinolones. Variations in DNA repair may be a significant risk factor for several cancers, but its measurement in epidemiological studies has been hindered by the high variability, complexity and laborious nature of currently available assays. An alkaline unwinding flow cytometric assay using UV-C radiation as a DNA-damaging agent was adapted for measurement of NER-mediated breaks. This assay was based on the principle of alkaline unwinding of strand breaks in double-stranded DNA to yield single-stranded DNA with the number of strand breaks being proportional to the amount of DNA damage. This assay measured 50,000 events per sample with several samples being analyzed per specimen, thereby providing very reliable measurements, which can be performed on a large-scale basis. Using area under the curve (AUC) to quantitate amount of NER-mediated breaks, this assay was able to detect increased NER-mediated breaks with increasing doses of UV-C radiation. The assay detected NER-mediated breaks in lymphocytes from normal donors and not in xeroderma pigmentosum lymphoblastoid cell lines indicating specificity for the detection of NER-mediated breaks. The assay measured NER-mediated breaks within G(1), S and G(2)/M phases of the cell cycle; thereby decreasing variability in measurements of NER-mediated breaks due to differences in cell cycle phases. Intraindividual variability for AUC after 120 min of repair was 15% with interindividual variability being approximately 43% for cells in the G(1) phase, indicating substantial between-subject variation and relatively low within-subject variation. Thus, the alkaline unwinding flow cytometry-based assay provides a high-throughput method for the specific measurement of NER-mediated breaks in lymphocytes.     

抑癌基因KLF6在肝癌HepG2细胞修复DNA损伤过程中的作用研究

目的：研究抑癌基因KLF6对肝癌HepG2细胞修复DNA损伤能力的影响。 方法: RT-PCR及Western blotting技术测定HepG2细胞在5 mg/L顺铂作用12、24、36 h后KLF6 mRNA及其蛋白水平变化。采用宿主细胞再活化反应(HCR)检测KLF6稳定表达HepG2细胞修复DNA损伤的能力。结果： 在5 mg/L顺铂作用下,随作用时间延长,KLF6 mRNA及其蛋白水平增高,但作用36 h,KLF6 蛋白水平下降。KLF6稳定表达HepG2细胞修复被顺铂损伤质粒DNA的能力明显高于对照组。结论： DNA损伤能诱导肝癌HepG2细胞KLF6基因表达,KLF6基因在细胞DNA损伤修复过程中发挥一定作用。