US20060147929A1 - Method for the quantitative assessment of global and specific dna repair capacities of at least one biological medium, and the applications thereof - Google Patents

Method for the quantitative assessment of global and specific dna repair capacities of at least one biological medium, and the applications thereof Download PDF

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US20060147929A1
US20060147929A1 US10/539,769 US53976905A US2006147929A1 US 20060147929 A1 US20060147929 A1 US 20060147929A1 US 53976905 A US53976905 A US 53976905A US 2006147929 A1 US2006147929 A1 US 2006147929A1
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plasmids
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Sylvie Sauvaigo
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Commissariat a lEnergie Atomique et aux Energies Alternatives CEA
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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  • the present invention relates to a method for the quantitative assessment of the overall and specific DNA repair capacities of a biological medium, by assessing the excision/resynthesis capacities of said medium, and also to the applications thereof.
  • DNA is continuously subjected to endogenous or exogenous attacks that result in the formation of base or sugar lesions.
  • the lesions include:
  • lesions of purine or pyrimidine bases oxidative lesions induced by the cellular metabolism and by photosensitization; lesions through the formation of chemical adducts, which result from the harmful action of many genotoxic agents, such as polycyclic hydrocarbons, contained in combustion products; lesions through the formation of metheno-bases or of etheno-bases;
  • lesions of the structure of the DNA double helix formation of intrastrand (between two adjacent bases of the same strand) or interstrand (between two bases located on the homologous strands) bridges, generally caused by ultraviolet radiation (formation of bridges between pyrimidines, which become dimeric) or bifunctional antitumor agents, such as cisplatin and intercalating agents, which form stable covalent bonds between the bases carried by opposite strands;
  • lesions through single-stranded or double-stranded breakage produced by agents such as ionizing radiation and through the action of free radicals;
  • the diversity of the induced lesions is illustrated by analyzing the stable photoproducts detected following UVC irradiation: alongside the pyrimidine dimers due to the formation of a cyclobutane ring, pyrimidine (6-4) pyrimidones form between two adjacent pyrimidines.
  • the relative proportion of the pyrimidine dimers and (6-4) products ranges from 10 to 4 for 1.
  • Their respective effectiveness in the lethal effect and in the mutagenic effect of ultraviolet radiation is also different: the dimers have a greater cytotoxic role than the (6-4) products, whereas the reverse is true for the mutagenic effect.
  • ionizing radiation ⁇ -rays from cobalt 60, for example
  • single-stranded or double-stranded breakages approximately in the ratio of 9 to 1
  • base addition products base losses
  • base losses base losses
  • bridges between DNA and adjacent proteins chromosomal, for example.
  • one strand breakage is counted per modified base.
  • the predominant role of double-stranded breakage in the cytotoxic effect of radiation is accompanied by a mutagenic effect due to base alterations.
  • modified DNA Another means of preparing modified DNA consists in manipulating plasmids by means of molecular biology techniques and inserting therein an oligonucleotide obtained by chemical synthesis and containing a lesion of interest (Biade et al., J. Biol. Chem., 1997, 273, 898-902).
  • BER base excision repair
  • NER nucleotide excision repair
  • the BER system is more specifically dedicated to the repair of small lesions in DNA, such as oxidative damage, abasic sites, base fragmentations, base methylations, etheno-bases, etc.
  • the NER system takes care of bulky lesions that induce distortion of the DNA double helix, such as acetylaminofluorine-DNA, cisplatin-DNA and psoralen-DNA adducts, dimers derived from UVB and UVC irradiation of DNA, covalent lesions formed between a DNA base and another molecule, etc. (Sancar et al., Annu. Rev. Genetics, 1995, 29, 69-105).
  • the repair generally ends with ligation of the neoformed strand with the existing DNA strand.
  • this process comprises the elimination of the modified nucleotide and the incorporation, as a replacement in the DNA chain, of at least one nucleotide triphosphate present in the repair medium.
  • repair systems especially in eukaryotes, are very complex and many variants of this simplistic configuration exist (overall repair, repair associated with the DNA transcription, repair associated with DNA replication, etc.).
  • Some proteins are involved in several repair systems simultaneously, others are specific for a single system, some can be induced by cellular or external factors, others have a ubiquitous and constant expression.
  • substrate refers to any DNA that may undergo a repair reaction in the presence of cell extracts and, by extension, the DNA lesions.
  • biological medium or “cell extract” refers to a purified by unpurified biological preparation that may contain at least one enzyme activity related to DNA repair.
  • a lesion can generally be associated with the specific proteins responsible for its repair in the DNA. Differences exist according to species; in prokaryotes such as Escherichia coli, the enzymes are less specific, whereas in humans, a much stricter lesion-specific repair enzyme association is observed, especially in the BER system. For example, Lindahl and Wood (Science, 1999, 286, 1897-1905) describe the BER system enzymes that are the most important in humans and also the lesions that are associated therewith. For example, in humans, the OGG1 protein, which is a glycosylase belonging to the BER system, is associated with the repair of 8-oxo-2′-deoxyguanosine.
  • Cellular repair capacity assays have been developed and can be classified in two categories: in vitro assays that require the use of active cell extracts, and in vivo or semi in vivo systems carried out on live cells.
  • this assay comprises the use of a plasmid DNA into which lesions are introduced (by UV irradiation: formation of pyrimidine dimers, bridges; through the action of DNase I: single-stranded cleavage or breakage); the DNA thus modified is incubated at 30° C. in the presence of a repair preparation comprising at least: the cell extract to be assessed, a nucleotide triphosphate labeled in the alpha-position with 32 P and ATP.
  • the enzymes contained in the extract incise the plasmid DNA and eliminate the lesions. DNA is synthesized de novo by replacement of the eliminated nucleotides.
  • the radioactive nucleotide introduced into the medium is incorporated into the DNA during the synthesis.
  • the amount of radioactivity incorporated which is proportional to the rate of repair of the substrate, is measured.
  • the method of preparing the cell extract and the reaction conditions influence the quality of the repair. In particular, it appears that the best repair yield is obtained with whole cell extracts of the type of those used for in vitro transcriptions, whereas cytosolic extracts of the type of those used for promoting plasmid replication from an SV40 origin, and also other crude cell extracts, exhibit nuclease activity, which does not allow correct interpretation of the repair.
  • the specificity of the reaction as regards the irradiated DNA is greater in the presence of a KCl concentration of the order of 40-100 mM.
  • the irradiated DNA replication which takes place during repair, is highly dependent on the presence of ATP and of an ATP-regenerating system (phosphocreatine+creatine phosphokinase), with a view to maintaining a constant level of ATP, the maintaining of this level being more specifically associated with the incision step of the repair.
  • a dependency is not, for example, encountered in cases of strand breakage repair.
  • a control sample consisting of the same plasmid that has not been modified, is used simultaneously in the reaction mixtures.
  • Wood et al. has been proposed in assays characterizing extracts originating from cells established from patients suffering from xeroderma pigmentosum (Satoh et al., Proc. Natl. Acad. Sci. USA, 1993, 90, 6335-6339; Jones et al., Nucl. Acids Res., 1992, 20, 991-995; Robins et al., EMBO J., 1991, 10, 3913-3921).
  • Xeroderma pigmentosum is a multigenic, multiallelic, autosomal recessive disease. Cells originating from patients suffering from this disease are very sensitive to ultraviolet radiation and exhibit DNA repair deficiencies.
  • XPA to XPG Eight genes are involved in the various complementation groups of this disease: XPA to XPG and the variant group XPV.
  • Each group has different characteristics relating to DNA repair and in particular to the various NER subtypes. DNA lesions, whether they belong to the small lesion category or the bulky lesion category, are repaired differently, according to the complementation group.
  • the repair in the presence of a cell extract is carried out in a reaction medium of 50 ⁇ l, using an extract comprising 150 ⁇ g of proteins, 50 mM of KCl, 5 mM of magnesium chloride, DTT, phosphocreatine, phosphocreatine kinase and various dNTPs, one of them being labeled with digoxigenin.
  • the repair is obtained after incubation for 3 hours at 30° C.
  • a washing solution comprising a phosphate buffer with a salt, to which a nonionic surfactant (Tween 20) is added in a proportion of 0.05 and 0.15% (preferred composition: 10 mM phosphate buffer, 137 mM NaCl and 0.1% Tween 20). It is specified that this assay is highly sensitive, insofar as the detection is carried out on 40 ng of DNA instead of 200 or 300 ng, in the case of an assay solution.
  • a nonionic surfactant Tween 20
  • the assay by the team of B. Salles and P. Calsou essentially proposes modifying the plasmid after attachment to the solid support.
  • chain breakages included among the lesions created by many chemical or physical agents.
  • breakages are repaired very rapidly and effectively by active cell extracts.
  • Breakage repair can even mask the repair of other lesions and interfere with the signals attributed to the repair of other DNA lesions. It is therefore a system that allows the detection of an overall effect, without identifying the lesions recognized by the repair systems; in addition, the aim of this method is not to detect and quantify the activity of the proteins involved in the DNA repair, but to identify the presence of lesions on the DNA processed.
  • A. Redaelli et al. (Terat. Carcinog. Mut., 1998, 18, 17-26) describe a method in which the plasmid is incubated directly with the extract without the nucleotide triphosphates. Cleavages in the supercoiled plasmid bring about a change in migration rate in the agarose gel during electrophoresis. The supercoiled plasmid migrates faster that the incised plasmid, due to its conformation. The bands corresponding to the various forms of the plasmid are quantified; the amount of the incised form is correlated with the activity of incision of the lesions of the plasmid, containing the extract.
  • this article studies the incision action of AP-endonuclease, that occurs on an a basic site, obtained after the action of a glycosylase specific for the modification to be repaired (alkylation, hydrolytic deamination, oxidation, mismatching), by cleaving the deoxyribose phosphodiester linkage positioned 3′ or 5′ of this abasic site.
  • the AP-endonuclease activity is more specifically studied on a crude extract of human lymphocytes. The extract (80 ⁇ l) is incubated, firstly, with an undamaged plasmid (control) and, secondly, with a depurinated plasmid. It is thus found to be possible to quantify the activity of AP-endonuclease insofar as the incision activity is dependent on the damage and sensitive to EDTA.
  • the presence of cleavage induces more rapid migration of the DNA than the nucleoid as a whole.
  • the ball of DNA then has the appearance of a comet, the intact DNA being in the head and the DNA containing cleavages being in the tail of the comet.
  • the percentage of DNA in the tail of the comet correlates directly with the incision activity contained in the extracts used for the lesions under consideration. This assay has been applied to measuring activities of excision of oxidative damage in extracts originating from human lymphocytes. Compared with the method described by Redaelli et al., 1998, which measures the cleavage of plasmids, the method of Collins et al.
  • this method comprises the attachment to a solid support of at least one damaged DNA containing at least one known lesion; this damaged DNA is then subjected to the action of a repair composition containing or not containing at least one protein involved in the repair of this damaged DNA, and the determination of the activity of this protein for the repair by measuring the variation of a signal emitted by a label that attaches to or removes itself from the support during the preceding step.
  • This system which is used with a damaged DNA that is in the form of an oligonucleotide of 15 to 100 bases or of a polynucleotide of 100 to 20 000 bases, thus makes it possible to obtain a more overall piece of information than the other assays, since the excision of several substrates can be monitored simultaneously.
  • this method concerns the demonstration of DNA lesion incision activities. It is thus limited to characterizing the step of excision of lesions that may be introduced into synthetic oligonucleotides. Furthermore, although it provides considerable information regarding excision activities, it is not suitable for and does not describe a precise quantification of the enzyme activities for excision/resynthesis of DNA.
  • the assays described above require the use of amounts of biological material and in particular of cell extracts of greater than 10 ⁇ l: the reaction volume generally used is 50 ⁇ l containing from 10 to 40 ⁇ l of extract for an amount of proteins of approximately 100 ⁇ g.
  • the extracts take a long time to prepare, and the amount of available cells is often small, which limits the number of assays that can be carried out.
  • each assay is carried out individually in a tube, i.e. a reaction takes place in the presence of a given plasmid and of a given extract.
  • the rate of incorporation of the label into the plasmid is compared with the rate of incorporation of the label obtained in a substrate prepared in an identical manner in the presence of the control extract.
  • the reference control extract is generally prepared from characterized cells transformed with EBV or SV40. The same is true in most of the other variants of the method by Wood et al., described above.
  • the lesions introduced into the plasmids are neither measured nor quantified.
  • the authors using the assay developed by Wood et al., eliminate only the plasmids that have lost their supercoiled form, in order to eliminate DNA containing chain breakages.
  • the information obtained is very partial and insufficient to precisely define and characterize the repair capacities of a given biological medium.
  • a subject of the present invention is a method for the quantitative assessment of the overall and specific DNA repair capacities of at least one biological medium, which method is characterized in that it comprises the following steps:
  • step (d) incubating said functionalized support obtained in step (c) with various repair solutions, each comprising at least one biological medium that may contain enzyme activities for repair, ATP, an ATP-regenerating system, a labeled nucleotide triphosphate and any other component necessary for the activity of the repair enzymes present in said biological medium, preferably at a temperature of 30° C. for 1 to 5 hours, preferably for 3 hours, each of said repair solutions being deposited, prior to said incubation, in each of said different and pre-established zones A 1 to A x of said functionalized support,
  • step (f) directly or indirectly measuring the signal produced by the label incorporated into the DNA during the repair reaction in step (d), in each of said different and pre-established zones A 1 to A x ,
  • the method according to the invention makes it possible to “map” a given biological medium in terms of its enzyme activities for DNA repair. It makes it possible to identify a biological extract according to the map obtained.
  • the method according to the invention also makes it possible to compare the performance levels of various biological extracts in terms of DNA lesion repair.
  • the plasmids prepared in step (a) are chosen from those that have a double-stranded supercoiled form (pBR322, M13, pUC, etc).
  • the supercoiled form of the control plasmid is obtained by purification using known techniques, for instance the Qiagen plasmid purification kits. It is also preferable to limit the presence of unwanted plasmid forms by carrying out other purification steps, for instance cesium chloride centrifugation and/or sucrose gradient centrifugation.
  • the various physical, biological or chemical agents capable of inducing a lesion of the DNA are chosen from those that preferably induce: the formation of a single lesion, the formation of a limited number of lesions or the formation of various lesions belonging to the same family.
  • oxidative lesions As families of lesions, mention may, for example, be made of: oxidative lesions, photoproducts induced by ultraviolet B or C radiation, chemical adducts, etheno-bases, abasic sites and DNA breakages.
  • the physical and chemical agents are, for example, chosen from those that function mainly:
  • the main target of singlet oxygen is guanine; in this case, the abundant lesion formed is 8-oxoguanine (Ravanat et al., Chem. Res. Tox., 1995, 8, 379-388);
  • the DNA lesions obtained are oxidative lesions; these lesions affect the purine bases and the pyrimidine bases in the DNA in an equivalent manner.
  • the main lesions formed are cyclobutane pyrimidine dimers (Costalat et al., Photochem, Photobiol., 1990, 51, 255-262);
  • the linkages formed are cyclobutane pyrimidine dimers, (6-4) photoproducts and the Dewar valence isomer (Douki et al., J. Biol. Chem., 2000, 275, 11678-11685);
  • the lesion formed is, in this case, 8-oxoguanine (Ravanat et al., J. Biol. Chem., 2001, 276, 40601-40604).
  • the chemical agents are chosen from those that induce known base modifications, belonging, inter alia, to the carcinogen family. Mention may, for example, be made of: acetylaminofluorene (Hess et al., 1996, Nucleic Acid Res. 24, 824-828), cisplatin (Pasheva et al., 2002, Int. J. Biochem. Cell Biol., 34, 87-92), benzopyrene (Laws et al., 2001, Mut. Res.; 484, 3-18), psoralen (Zhang et al., Mol. Cell.
  • step (a) of said method various agents are used on each plasmid of said range of plasmids.
  • the characterization of the lesions comprises (i) taking a fraction of each plasmid with lesions, (ii) digesting each of said fractions with enzymes that release the nucleosides from the DNA, and then (iii) analyzing the result of the digestion using a combination of separative techniques coupled to a quantitative analytical technique.
  • the digestion is carried out using at least one of the following enzymes: calf spleen phosphodiesterase, P1 nuclease, snake venom phosphodiesterase, and alkaline phosphatase (Douki et al., J. Biol. Chem., 2000, 275, 11678-11685).
  • the result of the enzyme digestion is analyzed by means of one of the following techniques: high performance liquid chromatography (HPLC) coupled to tandem mass spectrometry (Douki et al., 2000, J. Biol. Chem., 275, 11678-11685; Sauvaigo et al., 2001, Photochem. Photobiol., 73, 230-237; Frelon et al., Chem. Res. Tox., 2000, 13, 1002-1010), by HPLC coupled to gas chromatography (Wang et al., 2000, 13, 1149-1157; Pouget et al., 2000, Chem. Res. Tox., 13, 541-549) or else by HPLC coupled to electrochemical detection (Pouget et al., 2000, Chem. Res. Tox., 13, 541-549).
  • HPLC high performance liquid chromatography
  • tandem mass spectrometry Douki et al., 2000, J. Biol. Chem., 275, 11678-
  • the supercoiled forms of the plasmid obtained in step (a) are purified, preferably by sucrose gradient centrifugation and/or cesium chloride gradient centrifugation.
  • each of the plasmids of the range of plasmids is diluted to a concentration of between 5 and 100 ⁇ g/ml, in a diluting buffer preferably comprising a buffer at a pH of between 6.5 and 8.0, optionally combined with a salt and with a nonionic surfactant; preferably, said buffer is a 10 mM phosphate buffer or an SSC buffer, that can contain 0.05M to 0.5M NaCl.
  • the various plasmids are preferably deposited using a robot intended for the production of microarrays, i.e., the volumes deposited are preferably between 100 and 1000 picoliters.
  • said support is a support that has been sensitized so as to increase its affinity for the DNA, selected from the group consisting of organic or inorganic materials chosen from glass, silicon and its derivatives, and synthetic or nonsynthetic polymers (nylon or nitrocellulose membranes), and the surface of which is optionally functionalized; preferably, said support consists of glass slides coated with poly-L-lysine that adsorb the DNA, or glass slides functionalized with epoxy groups that form covalent bonds with the DNA.
  • treatments are performed so as to increase the attachment of the DNA to its support. These treatments must not create additional lesions in the deposited DNA.
  • a standard support according to the invention comprising zones A 1 to A x , each zone comprising the entire range of plasmids, comprises, in each of said zones:
  • step (d) of the method according to the invention
  • the biological extract can be prepared from the biological medium, according to the method of Manley et al., 1983, Methods Enzymol. 101, 568-582 or according to the method of Biade et al., J. Biol. Chem., 1998, 273, 898-902, or according to any other method capable of providing a medium containing repair proteins;
  • the label is selected from affinity molecules, fluorescent compounds, antibodies or biotin; preferably, the label or agent for visualizing the label is in particular chosen from the group consisting of fluorescent compounds with direct fluorescence (Cy-3 or Cy-5) or indirect fluorescence (biotin or digoxigenin);
  • the support is then incubated at a temperature that promotes the repair reaction, preferably 30° C., for a period of between one and five hours, preferably for three hours.
  • the support is washed at least once with a saline solution containing a nonionic surfactant, in particular a 10 mM phosphate buffer, containing Tween 20, and is then subsequently rinsed with water at least once.
  • a nonionic surfactant in particular a 10 mM phosphate buffer, containing Tween 20
  • the signal is measured by means of a method suitable for the label; for example, if the label is a fluorophore, direct measurement of the fluorescent signals emitted by the various deposits on the support is carried out.
  • said signals are quantified using a device capable of exciting the label, preferably a fluorophore, and of measuring the signal emitted subsequent to the excitation.
  • the signal is measured by means of instrumentation suitable for the support and for the label used.
  • a scanner may be used for the fluorescence image analysis, preferably with laser excitations at the wavelength specific for the label used.
  • step (h) of the method according to the invention a numerical ratio of the signals obtained with the plasmids containing the lesions to the signal obtained with the control plasmid located on the same support is established.
  • This repair profile can be used to determine the overall and specific repair capacities of a medium, to diagnose a repair-related disease, or to assess the influence of a physical or chemical treatment (genotoxic product, for example) on the repair capacities of a given medium.
  • FIG. 1 illustrates an example of configuration of the solid support; a plan of deposition in nine zones is observed;
  • FIG. 2 represents the deposition plan of each zone
  • FIG. 3 represents a repair diagram—repair mapping associated with each cell line that was used to prepare the extract used for the repair reaction.
  • the plasmid pBluescript II is produced by transformation of XL1-Blue MRF supercompetent cells from Stratagene, according to the protocol provided by Stratagene.
  • the plasmid is then purified using the Qiagen plasmid midi kit, according to the recommended protocol.
  • the plasmid is loaded onto 10 ml of 5-20% sucrose gradient in a 25 mM Tris HCl buffer, pH 7.5; 1M NaCl; 5 mM EDTA and centrifuged in a Beckman ultracentrifuge using an SW-41 rotor, at 4° C. and at 25 000 rpm for 18 hours. 1 ml fractions are then carefully taken and analyzed on an agarose gel. Only the fractions containing at least 90% of coiled form of the plasmid are kept. The plasmid is precipitated with ethanol and dissolved in PBS.
  • the plasmid diluted to 20 ⁇ g/ml in a PBS, is irradiated using a Bioblock germicidal lamp equipped with two 15-Watt neons. Three plasmid preparations are irradiated, respectively, at 0.06; 0.12 and 0.2 J/cm 2 .
  • CCA-Signa Treatment with chloroacetaldehyde (CCA-Signa)—Formation of malondialdehyde-deoxyguanine (MDA-dG)
  • the plasmid is prepared at 1 mg/ml in PBS, and an equivalent volume of CAA (50% in H 2 O) is added. This solution is incubated overnight at 37° C. The plasmid is recovered by precipitation and purified on a sucrose gradient.
  • a fraction of plasmid DNA or of calf thymus DNA treated under the same conditions is taken for analysis of the modified base composition.
  • the DNA is digested as described by Douki et al., J. Biol. Chem., 275, 11678-11685, and the analysis is then carried out by HPLC-tandem mass spectrometry.
  • the plasmids are diluted to 20 ⁇ g/ml in PBS. 500-picoliter deposits are made, using a GESIM robot, on commercial poly-L-lysine-coated glass slides (VWR). The slides are conserved at 4° C.
  • Each slide (support S) comprises 9 identical zones (A1 to A9) arranged according to configuration A in FIG. 1 .
  • each zone the range of plasmids is deposited in accordance with FIG. 2 , which illustrates, for example, zone A1.
  • Each zone makes it possible to test a different biological medium.
  • a solution is prepared containing the biological medium or extract to be tested; for 5 ⁇ l of solution, the composition is as follows: extract 0.5 ⁇ l 5 ⁇ repair buffer 1 ⁇ l CY5-dUTP (Amersham Pharmacia Biotech) 0.2 ⁇ l (0.1 nmol/ ⁇ l) 2M KCl 0.2 ⁇ l ATP (Roche - 100 mM) 0.1 ⁇ l
  • the volume is made up to 5 ⁇ l with H 2 O.
  • Hepes/KOH 200 mM, pH 7.8; 35 mM MgCl 2 ; 2.5 mM DTT; 2 ⁇ M dATP, 2 ⁇ M dGTP; 2 ⁇ M dCTP; 50 mM phosphocreatine; 250 ⁇ g/ml creatine phosphokinase; 0.5 mg/ml BSA; 17% glycerol.
  • the example is performed with three different extracts originating from different cell lines:
  • Line 1 these are HeLa cells.
  • the extracts are commercial nuclear extracts and come from the company 4C Biotech (Belgium). They were prepared by the method of Dignam et al., (Nucl. Ac. Res., 1983, 11, 1475-1489). Their protein content is 24 mg/ml.
  • Line 2 this is a line of AS203 cells established from a patient suffering from xeroderma pigmentosum, complementation group D.
  • the extracts were prepared according to the protocol of Manley et al. Assaying of proteins using the micro BCA kit makes it possible to evaluate the amount of proteins at 44 mg/ml.
  • Line 3 these are XP12RO cells. This line was established from a patent suffering from xeroderma pigmentosum, complementation group A. The extracts were prepared according to the protocol of Manley et al., (Methods Enzymol., 1983, 101, 568-582). The extract obtained contains 36 mg/ml of proteins (micro BCA assay kit, Interchim).
  • the fluorescence of the various deposits of each zone is analyzed by means of an Axon scanner at 635 nm and the GenePix Pro analytical software. A mean of three identical points is then determined. A value is thus obtained for each type of modification.
  • a diagram is plotted for each cell line. This diagram corresponds to a mapping of the repair systems associated with the lesions present on the support or chip, and is specific for the extract used. The results obtained are given in FIG. 3 .
  • the level of fluorescence is given in arbitrary units (AU).
  • each diagram is unique and specific for the cell line that was used to prepare the cell extract used. It can therefore be used to precisely characterize the overall repair activities of a given cell extract, and reveals the functionality of the systems targeted.
  • HeLa line repairs lesions induced by DDE (predominantly etheno-dG) twice as effectively as it does lesions induced by UV radiation (predominantly CPD and (6-4)). It is observed that the oxidative damage (predominantly 8-oxo-dG) is repaired much more weakly.
  • the XP12RO line it is observed that the DDE-induced lesions (predominantly etheno-dG) are the most effectively repaired, giving a signal three times higher than in the case of UVC radiation. It is known that the XPA lines do not repair CPDs; the signals obtained with UVC-irradiated DNA can thus be attributed to the repair of (6-4) photoproducts.
  • An unexpected advantage of the invention is that, even if the amount of protein is different from one extract to the other, the ratio obtained for the signals of the DNAs comprising the lesions to the signal of the control DNA, for a given extract, can be used to compare the repair capacities of the various extracts with respect to one another.

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WO2013035071A1 (en) 2011-09-08 2013-03-14 Yeda Research And Development Co. Ltd. Novel risk biomarkers for lung cancer
WO2021028909A1 (en) 2019-08-12 2021-02-18 Yeda Research And Development Co. Ltd. Dna repair blood test for predicting response of lung cancer patients to immunotherapy

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FR2887261B1 (fr) 2005-06-20 2007-09-14 Commissariat Energie Atomique Procede d'immobilisation de l'adn superenroule et utilisation pour analyser la reparation de l'adn
CN102031285B (zh) * 2009-09-28 2016-12-21 复旦大学 一种基于双核微核的dna修复能力检测方法
US10197578B2 (en) 2014-02-17 2019-02-05 Universite Claude Bernard Lyon 1 Predictive method of characterizing the radiosensitivity and tissular reaction of a patient to therapeutic ionizing radiation
FR3078343A1 (fr) * 2018-02-27 2019-08-30 Lxrepair Methode pour generer un profil des capacites de reparation de l'adn de cellules tumorales et ses applications
CN110208405A (zh) * 2019-05-30 2019-09-06 江苏恒生检测有限公司 一种检测水稻上呋虫胺残留的方法
WO2022184907A1 (en) 2021-03-04 2022-09-09 Lxrepair Multiplex quantitative assay for dna double-strand break repair activities in a biological medium and its applications

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US20070269824A1 (en) * 2006-03-02 2007-11-22 Jeffrey Albrecht Methods and systems for evaluating health risk factors by measurement of DNA damage and DNA repair
WO2013035071A1 (en) 2011-09-08 2013-03-14 Yeda Research And Development Co. Ltd. Novel risk biomarkers for lung cancer
WO2021028909A1 (en) 2019-08-12 2021-02-18 Yeda Research And Development Co. Ltd. Dna repair blood test for predicting response of lung cancer patients to immunotherapy

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KR101123976B1 (ko) 2012-03-23
JP2006510385A (ja) 2006-03-30
FR2849058A1 (fr) 2004-06-25
US9617580B2 (en) 2017-04-11
AU2003299364A1 (en) 2004-07-22
US20150031580A1 (en) 2015-01-29
DE60309930D1 (de) 2007-01-04
FR2849058B1 (fr) 2005-02-25
DE60309930T2 (de) 2007-09-20
ES2276157T3 (es) 2007-06-16
PL384058A1 (pl) 2008-06-09
ATE346171T1 (de) 2006-12-15
WO2004059004A3 (fr) 2004-08-19
EP1576191B1 (de) 2006-11-22
EP1576191A2 (de) 2005-09-21
WO2004059004A2 (fr) 2004-07-15
CN1738913A (zh) 2006-02-22

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