Constrained G4 structures unveil topology specificity of known and new G4 binding proteins.

G-quadruplexes (G4) are non-canonical secondary structures consisting in stacked tetrads of hydrogen-bonded guanines bases. An essential feature of G4 is their intrinsic polymorphic nature, which is characterized by the equilibrium between several conformations (also called topologies) and the presence of different types of loops with variable lengths. In cells, G4 functions rely on protein or enzymatic factors that recognize and promote or resolve these structures. In order to characterize new G4-dependent mechanisms, extensive researches aimed at identifying new G4 binding proteins. Using G-rich single-stranded oligonucleotides that adopt non-controlled G4 conformations, a large number of G4-binding proteins have been identified in vitro, but their specificity towards G4 topology remained unknown. Constrained G4 structures are biomolecular objects based on the use of a rigid cyclic peptide scaffold as a template for directing the intramolecular assembly of the anchored oligonucleotides into a single and stabilized G4 topology. Here, using various constrained RNA or DNA G4 as baits in human cell extracts, we establish the topology preference of several well-known G4-interacting factors. Moreover, we identify new G4-interacting proteins such as the NELF complex involved in the RNA-Pol II pausing mechanism, and we show that it impacts the clastogenic effect of the G4-ligand pyridostatin.

DNA replication but not nucleotide excision repair is required for UVC-induced replication protein A phosphorylation in mammalian cells.

Exposure of mammalian cells to short-wavelength light (UVC) triggers a global response which can either counteract the deleterious effect of DNA damage by enabling DNA repair or lead to apoptosis. Several stress-activated protein kinases participate in this response, making phosphorylation a strong candidate for being involved in regulating the cellular damage response. One factor that is phosphorylated in a UVC-dependent manner is the 32-kDa subunit of the single-stranded DNA-binding replication protein A (RPA32). RPA is required for major cellular processes like DNA replication, and removal of DNA damage by nucleotide excision repair (NER). In this study we examined the signal which triggers RPA32 hyperphosphorylation following UVC irradiation in human cells. Hyperphosphorylation of RPA was observed in cells from patients with either NER or transcription-coupled repair (TCR) deficiency (A, C, and G complementation groups of xeroderma pigmentosum and A and B groups of Cockayne syndrome, respectively). This exclude both NER intermediates and TCR as essential signals for RPA hyperphosphorylation. However, we have observed that UV-sensitive cells deficient in NER and TCR require lower doses of UV irradiation to induce RPA32 hyperphosphorylation than normal cells, indicating that persistent unrepaired lesions contribute to RPA phosphorylation. Finally, the results of UVC irradiation experiments on nonreplicating cells and S-phase-synchronized cells emphasize a major role for DNA replication arrest in the presence of UVC lesions in RPA UVC-induced hyperphosphorylation in mammalian cells.

A DNA double-strand break defective fibroblast cell line (180BR) derived from a radiosensitive patient represents a new mutant phenotype.

The 180BR cell line was derived from an acute lymphoblastic leukemia patient who overresponded to radiation therapy and died following radiation morbidity. 180BR cells are hypersensitive to the lethal effects of ionizing radiation and are defective in the repair of DNA double-strand breaks (DSBs). The levels and activity of the proteins of the DNA-dependent protein kinase complex are normal in 180BR cells. To facilitate a measurement of V(D)J recombination, we have characterized 180BRM, a SV40-transformed line derived from 180BR. 180BRM retains the radiosensitivity and defect in DSB repair characteristic of 180BR. The activities associated with DNA-dependent protein kinase are also normal in 180BRM cells. The ability to carry out V(D)J recombination is comparable in 180BRM and a reference control transformed human cell line, MRC5V1. These results show that 180BR and 180BRM differ from the rodent mutants belonging to ionizing radiation complementation groups 4, 5, 6, and 7 and, therefore, represent a new mutant phenotype, in which a defect in DNA DSB rejoining is not associated with defective V(D)J recombination. Furthermore, we have shown that 180BR can arrest at the G1-S and G2-M cell cycle checkpoints after irradiation. These results confirm that 180BR can be distinguished from ataxia telangiectasia.

Human normal peripheral blood B-lymphocytes are deficient in DNA-dependent protein kinase activity due to the expression of a variant form of the Ku86 protein.

The heterodimeric Ku protein, which comprises a 86 kDa (Ku86) amd a 70 kDa (Ku70) subunits, is an abundant nuclear DNA-binding protein which binds in vitro to DNA termini without sequence specificity. Ku is the DNA-targeting component of the large catalytic sub-unit of the DNA-dependent protein kinase complex (DNA-PK[CS]), that plays a critical role in mammalian double-strand break repair and lymphoid V(D)J recombination. By using electrophoretic mobility shift assays, we demonstrated that in addition to the major Ku x DNA complex usually detected in cell line extracts, a second complex with faster electrophoretic mobility was observed in normal peripheral blood lymphocytes (PBL) extracts. The presence of this faster migrating complex was restricted to B cells among the circulating lymphocyte population. Western blot analysis revealed that B cells express a variant form of the Ku86 protein with an apparent molecular weight of 69 kDa, and not the 86 kDa- full-length protein. Although the heterodimer Ku70/variant-Ku86 binds to DNA-ends, this altered form of the Ku heterodimer has a decreased ability to recruit the catalytic component of the complex, DNA-PK(CS), which contributes to an absence of detectable DNA-PK activity in B cells. These data provide a molecular basis for the increased sensitivity of B cells to ionizing radiation and identify a new mechanism of regulation of DNA-PK activity that operates in vivo.

Inhibition of Ku heterodimer DNA end binding activity during granulocytic differentiation of human promyelocytic cell lines.

The heterodimeric Ku protein (composed of the Ku 86 and Ku 70 sub-units) is a nuclear protein which binds to DNA termini without sequence specificity. Ku is the DNA-targeting component of the large catalytic sub-unit of the DNA-dependent protein kinase complex that is required for the repair of DNA double-strand breaks in mammalian cells. We studied the expression and function of Ku/DNA-PK during granulocytic differentiation of two human promyelocytic cell lines, HL60 and NB4, a process associated to decreased radiation resistance. After 3 days exposure to differentiating agents (either all-trans-retinoic acid or DMSO), Ku binding to double stranded (ds)-DNA ends declined dramatically whereas Ku protein levels remain unchanged. The nuclear, but not cytoplasmic, fraction of differentiated HL60 cells extracts exhibited a heat-sensitive inhibitory activity towards DNA binding of recombinant Ku heterodimer. We further demonstrate that immunoprecipitation of Ku is impaired in extracts from differentiated cells by using two antibodies that recognize epitopes within the C-terminus DNA binding domains of Ku 70 and Ku 86 proteins. These results favor the hypothesis of a protein interacting with Ku that would prevent DNA binding of heterodimerized Ku protein by steric hindrance.

Interactions of the transcription/DNA repair factor TFIIH and XP repair proteins with DNA lesions in a cell-free repair assay.

We have studied the interactions between DNA damage and human proteins involved in the early steps of nucleotide excision repair (NER) reaction under in vitro conditions with human protein extracts. By using a new assay, we have detected a long-lived DNA/protein complex involving XPA and TFIIH in the course of the NER process. The formation of this complex is exclusively limited to DNA lesions that are substrates of the human excinuclease. We show that, while XPA binding to damaged DNA is ATP-independent, stable association of TFIIH with DNA lesions is promoted by ATP hydrolysis and is dependent on the integrity of XPA and XPC proteins in the cell extract. In addition, XPC is necessary to promote a stable binding of XPA to UV-irradiated DNA. Finally, the co-binding of XPA and TFIIH to DNA damage is correlated to a dose-dependent titration of TFIIH and not XPA from the free protein fraction.

Ku70/Ku80 protein complex inhibits the binding of nucleotide excision repair proteins on linear DNA in vitro.

We have previously reported that the incision efficiency of the nucleotide excision repair (NER) reaction measured in vitro with cell-free human protein extracts was reduced by up to 80% on a linearized damaged plasmid DNA substrate when compared to supercoiled damaged DNA. The inhibition stemed from the presence of the DNA-end binding Ku70/Ku80 heterodimer which is the regulatory subunit of the DNA-dependent protein kinase (DNA-PK). Here, the origin of the repair inhibition was assessed by a new in vitro assay in which circular or linear plasmid DNA, damaged or undamaged, was quantitatively adsorbed on sensitized microplate wells. The binding of two NER proteins, XPA and p62-TFIIH, indispensable for the incision step of the reaction, was quantified either directly in an ELISA-like reaction in the wells with specific antibodies or in Western blotting experiments on the DNA-bound fraction. We report a dramatic inhibition of XPA and p62-TFIIH association with UVC photoproducts on linear DNA. XPA and p62-TFIIH binding to DNA damage was regained when the reaction was performed with extracts lacking Ku activity (extracts from xrs6 rodent cells) whereas addition of purified human Ku complex to these extracts restored the inhibition. Despite the fact that DNA-PK was active during the NER reaction, the mechanism of inhibition relied on the sole Ku complex, since mutant protein extracts lacking the catalytic DNA-PK subunit (extracts from the human M059J glioma cells) exhibited a strong binding inhibition of XPA and p62-TFIIH proteins on linear damaged DNA, identical to the inhibition observed with the DNA-PK+ control extracts (from M059K cells).

DNA repair activity in protein extracts from rat tissues.

Among DNA repair pathways, nucleotide excision repair (NER) is able to recognize and process a wide variety of DNA lesions. The NER mechanism can be summarized in two stages: incision/excision of the lesion and DNA repair synthesis. Here, we have assessed the repair synthesis activity of protein extracts from different rat tissues by an in vitro biochemical assay that reproduces the entire NER reaction. The protein extraction procedure was adapted to rat tissues and the biochemical parameters of the assay (high salt concentration, addition of EGTA) in order to minimize non-specific nuclease activity which allows the measurement of repair activity. Using this repair assay we detected a small increase in the extent of repair synthesis in liver compared to brain and lung tissue protein extracts. Similar results were obtained using a derivative assay that allows the measurement of the incision activity of tissue protein extracts with lower incision activity in lung tissue extract.

Transfer of Ku86 RNA antisense decreases the radioresistance of human fibroblasts.

Ku86 has been shown to be involved in DNA double-strand break (DSB) repair and radiosensitivity in rodents, but its role in human cells is still under investigation. The purpose of this study was to evaluate the radiosensitivity and DSB repair after transfection of a Ku86-antisense in a human fibroblast cell line. Simian virus 40-transformed MRC5V1 human fibroblasts were transfected with a vector (pcDNA3) containing a Ku86-antisense cDNA. The main endpoints were Ku86 protein level, Ku DNA end-binding and DNA protein kinase activity, clonogenic survival, and DSB repair kinetics. After transfection of the Ku86-antisense, decreased Ku86 protein expression, Ku DNA end-binding activity, and DNA protein kinase activity were observed in the uncloned cellular population. The fibroblasts transfected with the Ku86-antisense showed also a radiosensitive phenotype, with a surviving fraction at 2 Gy of 0.29 compared with 0.75 for the control and 20% of unrepaired DSB observed at 24 hours after irradiation compared with 0% for the control. Several clones were also isolated with a decreased level of Ku86 protein, a surviving fraction at 2 Gy between 0.05 and 0.40, and 10-20% of unrepaired DSB at 24 hours. This study is the first to show the implication of Ku86 in DSB repair and in the radiosensitivity of human cells. This investigation strongly suggests that Ku86 could constitute an appealing target for combining gene therapy and radiation therapy.

RECA immunological assay as a tool to analyze the SOS response.

The content of RECA protein, one of the SOS genes product, was determined in a bacterial extract by a two site-radioimmunometric assay. The variation of the RECA concentration after induction by physical or chemical treatments was used as a probe to analyze the SOS response. Relationships between either the number or the nature of DNA lesions and the level of the relative amplification of RECA have been established. The modulation of the recA gene expression is discussed.

The activity of the DNA-dependent protein kinase (DNA-PK) complex is determinant in the cellular response to nitrogen mustards.

The DNA-dependent protein kinase plays a critical role in mammalian DNA double strand break (DSB) repair and in specialized recombination, such as lymphoid V(D)J recombination. Its regulatory subunit Ku (dimer of the Ku70 and Ku80 protein) binds to DNA and recruits the kinase catalytic sub-unit, DNA-PKcs. We show here that three different strains deficient in either the Ku80 (xrs-6) or DNA-PKcs (V-3, scid) component of DNA-PK are markedly sensitive (3.5- to 5-fold) to a group of DNA cross-linking agents, the nitrogen mustards (NMs) (melphalan and mechlorethamine) as compared to their parental cell line. Importantly, the level of hypersensitivity to these drugs was close to the level of hypersensitivity observed for radiomimetic agents that create DSBs in DNA (bleomycin and neocarzinostatin). In addition, sensitivity to NMs was restored to the parental level in the xrs-6 cell line stably transfected with the human Ku80 gene (xrs-6/Ku80), showing unequivocally that DNA-PK is involved in this phenotype. These results indicate that a function of the whole DNA-PK protein complex is involved in the cellular response to NMs and suggest that the repair of DNA interstrand cross-links induced in DNA by NMs involved a DNA-PK dependent pathway that shares common features with DNA DSBs repair.

Regulation of the DNA-dependent protein kinase (DNA-PK) activity in eukaryotic cells.

The DNA-dependent protein kinase (DNA-PK) is a trimeric nuclear serine/threonine protein kinase consisting of a large catalytic sub-unit and the Ku heterodimer that regulates kinase activity by its association with DNA. DNA-PK is a major component of the DNA double strand break repair apparatus, and cells deficient in one of its component are hypersensitive to ionizing radiation. DNA-PK is also required to lymphoid V(D)J recombination and its absence confers in mice a severe combined immunodeficiency phenotype. The purpose of this review is to summarize the current knowledge on the mechanisms that contribute to regulate DNA-PK activity in vivo or in vitro and relates them to the role of DNA-PK in cellular functions. Finally, the studies devoted to drug-inhibition of DNA-PK in order to enhance cancer therapy by DNA-damaging agents are presented.

In vitro eukaryotic DNA excision repair assays: an overview.

Great progress is being made in understanding the process of nucleotide excision repair (NER) in eukaryotes. Different lines of research have been developed, among them an in vitro assay with cell-free extracts has played a major role. This in vitro repair assay takes advantage of a cell-free system that can mediate DNA excision-repair by transcriptionally active protein extracts from mammalian cells incubated in the presence of two plasmids of different sizes, one damaged and the other undamaged as internal control. The extent of repair activity is determined by following the level of radiolabeled incorporation during the repair synthesis step consecutive to the excision of DNA lesions. We discuss the interest and drawbacks of this biochemical assay in light of the main results obtained. We report the modifications that we have undertaken in order to determine repair synthesis activity in a chemiluminescent-directed reaction as well as to assess incision activity in protein extracts.

DNA damage excision repair in microplate wells with chemiluminescence detection: development and perspectives.

The development of in vitro repair assays with human cell-free extracts led to new insights on the mechanism of excision of DNA damage which consists of incision/excision and repair synthesis/ligation. We have adapted the repair synthesis reaction with cells extracts incubated with damaged plasmid DNA performed in liquid phase to solid phase by DNA adsorption into microplate wells. Since cells extracts are repair competent in base excision and nucleotide excision repair, all types of substrate DNA lesions were detected with chemiluminescence measurement after incorporation of biotin-deoxynucleotide during the repair synthesis step. Derivatives of our initial 3D-assay (DNA damage detection) have been set up to: i) screen antioxidative compounds and NER inhibitors; ii) capture genomic DNA (3D(Cell)-assay) that allows detection of alkylated base and consequently determines the kinetics of the cellular repair; and iii) immunodetect the repair proteins in an ELISA reaction (3D(Rec)-assay). The 3D derived assays are presented and discussed.

Involvement of glutathione in cis-platinum toxicity in Escherichia coli K12.

Among the various biochemical functions assumed by the tripeptide glutathione (GSH), a role in cell protection against xenobiotics has been well established. In the case of resistance to cis-diamminedichloroplatinum(II) (CDDP) this role is controversial. CDDP reacts with nucleophiles and binds covalently to DNA, its ultimate target. We addressed the question of a putative role of GSH as a secondary non-essential target by using a bacterial model. With an Escherichia coli K12 mutant devoid of GSH, we found sensitivity to CDDP increased by a factor of two. It appeared that GSH protects bacteria at least by covalently trapping platinum before its binding to DNA since (i) lower binding of CDDP to DNA was found when GSH was present and (ii) the resistance still persisted in bacteria after treatment by the monofunctional derivative [Pt(dien)Cl]Cl. On the other hand, with a DNA repair defective mutant (lexA3), we found that other biochemical secondary target(s) might be involved in bacterial protection at low CDDP concentrations.

Multiple mechanisms of resistance to cisplatin toxicity in an Escherichia coli K12 mutant.

The mechanisms underlying cellular resistance to the antitumor drug cis-diamminedichloro-platinum(II) (CDDP) were studied in Escherichia coli K12. A bacterial strain (MC4100/DDP) was selected from the MC4100 wild-type strain after growth for four cycles in CDDP. MC4100/DDP bacteria showed a high level of resistance and exhibited various modifications including (1) a decrease in drug uptake and platinum/DNA binding which only partly contributed to resistance, (2) an increase in glutathione content not involved in the resistant phenotype, (3) an increase in DNA repair capacity. Resistance was unmodified by introducing a uvrA mutation which neutralizes the excision-repair pathway. In contrast, it was abolished by deletion of the recA gene which abolishes recombination and SOS repair but also by a mutation in the recA gene leading to RecA co-protease minus (no SOS induction). RecA protein was unchanged in MC4100/DDP but the expression of RecA-dependent gene(s) was required for CDDP resistance. The regulation of genes belonging to the SOS regulon was analysed in MC4100/DDP by monitoring the expression of sfiA and recA::lacZ gene fusions after UV irradiation. These gene fusions were derepressed faster and the optimal expression was obtained for a lower number of UV lesions in MC4100/DDP, suggesting a role of RecA co-protease activity in the mechanism of resistance to CDDP in this E. coli strain.

UV resistance of E. coli K-12 deficient in cAMP/CRP regulation.

Deletion of genes for adenylate cyclase (delta cya) or cAMP receptor protein (delta crp) in E. coli K-12 confers a phenotype that includes resistance to UV radiation (254 nm). Such mutations lead to UV resistance of uvr+, uvrA, lexA and recA strains which could partly be abolished by the addition of cAMP to delta cya but not to delta crp strain culture medium. This effect was not related to either inducibility of major DNA repair genes or growth rate of the bacteria. Enhanced survival was also observed for UV-irradiated lambda bacteriophage indicating that a repair mechanism of UV lesions was involved in this phenomenon.

Modification of deoxyribose-phosphate residues by extracts of ataxia telangiectasia cells.

The release of DNA 5'-terminal deoxyribose-phosphate residues from enzymatically incised apurinic/apyrimidinic sites by human cell extracts has been under investigation. During the course of these studies, we observed that ataxia telangiectasia cell extracts modify deoxyribose-phosphate (dRp) residues by converting them to an altered form, dRp-X, which shows altered chromatographic properties on HPLC analysis. The chemical nature of the adduct is as yet unknown, but dRp-X is stable to both heat and acid. The modification requires an enzymatic activity and a low-molecular weight co-factor. Extracts of normal cells contain a dialyzable inhibitor that suppresses the reaction occurring with ataxia telangiectasia cell extracts. Formation of dRp-X has been observed in 7 out of 7 ataxia telangiectasia lymphoblastoid lines which represent at least 3 genetic complementation groups. Similar modification of dRp did not occur with extracts of cells of normal origin, nor those representing Fanconi's anaemia, xeroderma pigmentosum, Bloom's syndrome, Werner's syndrome or Friedreich's ataxia.

Repair of oxidative DNA damage in vitro: a tool for screening antioxidative compounds.

Reactive oxygen species (ROS) provoke the formation of base DNA alterations that are processed by an excision step of the lesion followed by a repair synthesis and ligation step to restore the strand continuity. We have reported previously the detection of DNA adducts by an in vitro chemiluminescence DNA repair synthesis assay (Salles et al., 1995) which allows the measurement of repair synthesis by cell-free extracts in damaged plasmid DNA adsorbed on sensitized microplate wells. The 3D (DNA damage detection) assay was performed in the presence of biotin-dUTP which was incorporated during the repair synthesis step. The extent of repair synthesis was measured in an ELISA reaction with ExtrAvidin-horse radish peroxidase and chemiluminescence detection. The 3D assay allows detection of any type of base alterations including base oxidation. Interestingly, under controlled production of ROS a screening procedure of antioxidants might be carried out with the 3D assay. By taking advantage of plasmid DNA adsorption, oxidative base damage can be recognized by the Escherichia coli Fpg protein which was detected in an ELISA reaction with specific antibody and chemiluminescence measurement (4D assay). With the sceening procedure of antioxidative compounds in mind, the development of such assays and their drawbacks are discussed.

[DNA-dependent protein kinase: a major protein involved in the cellular response to ionizing radiation]. / La protéine kinase dépendante de l'ADN (DNA-PK): une protéine majeure de la réponse cellulaire aux radiations ionisantes.

DNA-dependent protein kinase (DNA-PK) is a DNA-activated nuclear serine/threonine protein kinase. DNA-PK consists of a regulatory sub-unit, the heterodimeric Ku protein (composed of a 70- and a 86-kDa subunit) which binds DNA ends and targets the catalytic sub-unit, DNA-PKcs to DNA strand breaks. DNA-PK plays a major role in the repair of double-strand breaks induced in DNA after exposure to ionizing radiation as shown by the extreme radiosensitivity of cells with mutations in Ku86, Ku70 or DNA-PKcs genes. Cells deficient in DNA-PK activity also exhibit hypersensitivity to genotoxic drugs such as cisplatin and nitrogen mustards. In the first part of this review, the current knowledge on the biochemical characteristics of DNA-PK, its mechanism of action in DNA repair and the phenotype of DNA-PK deficient cells is summarized. These results suggest that DNA-PK might play a role in the acquisition of a resistant phenotype of human tumors to radiotherapy, chemotherapy using genotoxic drugs or to both treatments. In the second part of this review, the studies devoted to inhibition of DNA-PK in order to enhance cancer therapy by DNA-damaging agents are presented.