WO1998037233A1 - Method for the isolation and/or assay of damaged dna and its application to screening of cytotoxic products acting on dna - Google Patents

Method for the isolation and/or assay of damaged dna and its application to screening of cytotoxic products acting on dna Download PDF

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WO1998037233A1
WO1998037233A1 PCT/FR1998/000339 FR9800339W WO9837233A1 WO 1998037233 A1 WO1998037233 A1 WO 1998037233A1 FR 9800339 W FR9800339 W FR 9800339W WO 9837233 A1 WO9837233 A1 WO 9837233A1
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dna
tbp
transcription
complex
sample
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PCT/FR1998/000339
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French (fr)
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Jean-Marc Egly
Frédéric COIN
Dino Moras
Jean-Paul Renaud
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Institut National De La Sante Et De La Recherche Medicale (Inserm)
Centre National De La Recherche Scientifique (Cnrs)
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Publication of WO1998037233A1 publication Critical patent/WO1998037233A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6804—Nucleic acid analysis using immunogens
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer

Definitions

  • the present invention relates to a method for detecting and / or assaying the presence of damaged DNA in a DNA sample and its application to the detection and / or screening of products liable to damage DNA.
  • the external aggressions that DNA undergoes can be of various origins. They are generally grouped into two categories: physical aggressions produced in particular by radiation, and; chemical attacks.
  • DNA damage is a phenomenon which occurs in particular in cancer treatments.
  • the anticancer drug cisplatin is considered to be one of the DNA damaging agents (Zambia and Lippard, 1995), its use in the treatment of certain cancers is essential. This is why, in what follows, the evaluation of DNA damage could make it possible to highlight the toxicity of an aggressor agent as well as to follow the progress of an anti-cancer therapy. especially. The response provided by cells to these attacks of various origins which lead to DNA damage, results in the establishment of the so-called DNA repair mechanism.
  • This very complex repair mechanism implements a number of proteins, some of which are also involved in cellular processes other than repair.
  • the transcription factor TFIIH is considered to be essential both for transcription and for the mechanism of nucleotide repair by excision (Svejstrup et al;, 1996, Hoeijmakers et al., 1996).
  • TBP TATA binding protein
  • the present invention relates to a method for detecting and / or assaying damaged DNA in a sample, characterized in that the importance of the fixation of TBP or of a similar product is evaluated on the DNA of the sample and the results are determined directly or relative to a control.
  • TBP is understood to mean both TBP in free form, for example a purified recombinant protein and in its complexed form, for example the TBP / THIID complex. In general, one can also consider using a product similar to TBP which retains its ability to fix damaged DNA.
  • dosage is meant both a qualitative and a quantitative dosage.
  • evaluation is intended to determine the fixation or non-fixation of the TBP, which is done directly if, for example, calibration curves are available, or else by comparison with a control corresponding to undamaged DNA or damaged DNA controls. As will be demonstrated below, the damaged DNA / TBP complex is stable enough to be identified and assayed and the extent of TBP binding is a good indication of the extent of damage to DNA.
  • the demonstration or the assay can be carried out either directly or indirectly.
  • the complex formed itself is used as the dosing element
  • the present invention relates to a method for detecting and / or assaying damaged DNA in a sample characterized in that the importance of the binding of TBP to the DNA is evaluated by determination of the transcription of a known undamaged DNA, in a medium ensuring transcription, transcription being ensured only by the presence in said medium of residual TBP not fixed on DNA.
  • TBP is attached to damaged DNA, it is not available to transcribe undamaged DNA.
  • the measurement of the transcription, and therefore of the transcribed RNA corresponding to the undamaged DNA can be carried out by all known methods, migration on gel for example, as will be explained in more detail in the examples below.
  • the method is carried out as follows: a) the sample is incubated with TBP or a similar product; b) after incubation, a known undamaged DNA sequence is added to the sample, under conditions ensuring transcription but without additional TBP; c) determining the transcription of this known DNA and; d) the variation in transcription of this known DNA is measured with respect to a normal transcription or by comparison with a control.
  • step a) is carried out under conditions which do not allow DNA repair and step b) is carried out in a medium which can consist of total cellular extracts, extracted from HeLa cells by example or of a reconstructed transcription system.
  • the presence of damaged DNA is detected and / or measured in a sample by a method in which said sample is placed in the presence of a reagent containing TBP or an analogous product and the extent of the DNA damage is evaluated by detecting and / or assaying the T3P / DNA complex formed, directly or in relation to a control.
  • the TBP / DNA complex can be demonstrated and / or determined by any suitable method.
  • DNA adopts a curved structure which presents similarities to form A of DNA.
  • the resulting enlargement and flattening of the small groove allows hydrophobic contacts and promotes the formation of the damaged TBP / DNA complex.
  • the methods according to the present invention make it possible to highlight not only the damage to the labeled DNA, but also its semi-quantitative or quantitative assay.
  • radioactive or non-radioactive reagents for example fluorescent
  • the use of radioactive or non-radioactive reagents makes it possible by measuring a band intensity to make a quantitative assay.
  • the interest of these assays is first of all, at the stage of industrial research, to allow the search for cytotoxic products having a particular mode of action of the "cisplatin" type.
  • the method according to the present invention can be used as a method for screening for cytotoxic products on DNA, in particular anticancer drugs, these being revealed either by the appearance of the TBP / DNA complex, or by the decrease in the transcription of DNA that has not been damaged.
  • the methods according to the present invention can also be used to test the possible toxicity of certain products, cosmetics, herbicides, pharmaceuticals, for example by regular sampling in industrial enclosures at risk.
  • the method according to the present invention can also be used for therapeutic monitoring, in particular in anticancer therapies. More specifically, to determine the cytotoxicity on the DNA of a given product, said product is reacted beforehand in an undamaged DNA sample, said sample then being free of any other products capable of damaging DNA and one proceeds according to the different variants envisaged according to the invention, the cytotoxicity of the product being established when damaged DNA is detected.
  • FIGS. 1 to 7 represent: FIG. 1: Part (A): Schematizes a competition / transcription experience.
  • An AdMLP transcription matrix is generated by digestion / restriction with EcoRI / SalI of the plasmid pUC309. It gives rise to an RNA transcript of 309 nucleotides which contains the promoter AdMLP (hatched part).
  • the F879 fragment is created by digestion / restriction of pUC309 with BamHI and Sspl. This fragment does not contain any promoter sequence.
  • Figure 2 Shows the results obtained in experiments of inhibition of transcription in vitro from the AdMLP gene in the presence of DNA damaged by UV rays or by cisplatin.
  • Figure 3 (A to D): Represents assays performed using the fixation of the complex on a cellulose membrane.
  • Figure 4 (A and B): Shows the results of the assays performed using the fixation of the complex on a nitrocellulose membrane in the presence of competing fragments.
  • the substrates used for in vitro transcription or for the membrane binding test are prepared by digestion / restriction either of plasmid DNA from pUC309 or from pSK. Plasmid pUC309 is prepared by ligation of a fragment
  • Figure 1B The linear fragments are purified on 1% agarose gel and Qiaex beads (Qiagen company).
  • the DNA is resuspended in a 50 mM Tris-HCl buffer (pH 7.9), containing 10% glycerol, ImM of EDTA, 0.5 mM of DTT and 50 mM of KCl for use in the transcription or setting experiments.
  • evidence of protein / DNA complex (EMSA; membrane retention).
  • the final competitor used was the "Bluescript" of
  • plasmid pSK (Stratagene).
  • the F879 fragment was UV-damaged with a UV-C lamp at a power of 0.1 m / cm 2 .
  • the plasmid pSK is treated with a solution of 0.1 mg / ml in water of cisplatin (II) dichloride diamine (Sigma) for 15 hours in the dark at 37 ° C. and with a cisplatin / nucleotide ratio of 0.005 (Hansson and ood, 1989).
  • CP (-) or CP (+) fragments are used which are used subsequently in the membrane binding tests by digestion / restriction of pSK, respectively intact or damaged by cisplatin, with the restriction enzyme Pvul followed by purification of the 1084 bp fragment on 1% agarose gels without BET.
  • AdMLP 64 mer
  • AdMLP complementary synthesized oligonucleotides corresponding to the regions -40 to +24 of the late major promoter of adenovirus 2 (AdMLP).
  • the 32-mer 5 '-TCTTCTTCTTCTTCTGTGCACTCTTCTTCTCT-3' probe containing a single GpTpG is reacted with cisplatin (Moggs et al., 1996). After ethanol precipitation, the presence of a 1,3-cisplatin d (GpTpG) bond was confirmed by analysis of the oligonucleotide on a 12% acrylamide gel, the modified oligonucleotide migrating more slowly than l intact oligonucleotide.
  • the resulting DNA probe (36 bp) used in the EMSA test is prepared by pairing the oligonucleotide cisplatin with its complementary undamaged oligonucleotide leaving a free 5 'portion at each end.
  • the DNA is completed with incorporation of P32 dATP, radioactively labeled with P32 dATP (3000-Ci / mmol) in the presence of the Klenow fragment of the DNA polymerase I of E. coli and then purified on sephadex G50 columns.
  • reaction media (20 ⁇ l) are prepared containing a 50 mM Tris / HCl buffer pH 7.9, 0.2 ng of a 36 bp DNA probe marked with P32 (10,000 cpm) 80 mM KCl , 5 mM of MgCl2, 0.1 mM of EDTA, 500 ng of BSA, 10- of glycerol, 0.5 mM DTT, 0.01% NP40, recombinant TBP. After 30 minutes of incubation at 25 ° C, the samples are adjusted to 20% glycerol and deposited on 4% polyacrylamide gel. Electrophoretic migration takes place in a 25m MTris / 19mM Glycine buffer at room temperature. The gels are dried and exposed to biomax films (Kodak).
  • EXAMPLE 1 Method using the "transcription defect" due to the formation of the TBP / DNA complex (FIG. 2).
  • AdMLP matrix 90 ng of the AdMLP matrix (EcoRI / SalI) are then added which should give rise to a specific transcript which is left to incubate for 15 minutes to promote the formation of the transcription pre-initiation complex.
  • nucleotides triphosphate ATP, GTP, UTP, including p32 CTP (400Ci / mmol), final reaction volume of 25 ⁇ l, transcription will remain and will continue for 45 min at 25 ° C.
  • RNAs are analyzed after electrophoresis on 5% acrylamide gel, by autoradiography and then quantified directly by counting on a phospholmage analyzer or indirectly by densitometric scanning of autoradiograms using a BioRad GS700 image densitometer The results of this assay are shown in Figure 2.
  • Part (A) The results of the transcription of AdMLP carried out with 30 ⁇ g of WCE in the presence of increasing amounts of DNA fragment F879 irradiated with UV (650 J / m2) (columns 3-8) or not irradiated ( columns 9-14).
  • the upper part of the figure represents an autoradiogram, the lower part represents the densitometric quantification of the autoradiogram (band of 309 nucleotides): the percentage of transcription from AdMLP is expressed as a function of the quantity of competing F879 DNA expressed in ng.
  • Part (B) The results of the transcription of AdMLP carried out with 20 ⁇ g of EC in the presence of increasing amounts of undamaged pSK (CP (-)) (columns 2-8) or damaged by cisplatin (CP (+)) (columns 9-15).
  • the upper part of the figure represents the production of transcripts from AdMLP (band of 309 nucleotides).
  • the lower part of the figure represents the same type of quantification as part (A).
  • EXAMPLE 2 Decrease in transcription in a reconstituted transcription system (RTS) (Fig. 3B and D).
  • a highly purified reconstituted transcription system which contains the transcription factors, TFIIA, TFIIB, the TFIID / TBP complex, TFIIE, TFIIF and RNA pol II.
  • This system which contains all the factors essential for transcription, is pre-incubated with DNA F879 damaged by irradiation with 500 (col. 6 to 9) or 1000 (col. 10 to 13) J / m 2 and tested for its ability to inhibit transcription from a matrix of the reporter gene AdMLP.
  • the presence of damaged DNA preferentially inhibits the production of the 309 nucleotide transcript (FIG. 3B), which is in agreement with the previous observations and the binding to the nitrocellulose membrane.
  • Fig 3D An inhibition is observed proportional to the quantity of damaged DNA and therefore to the amount of lesions due to UV (compare columns 2-5 with columns 6-9 and 10-13) or due to cisplatin (Fig 3D) (comparison columns 2 and 3 with columns 4 and 5), which demonstrates that the capacity of damaged DNA to inhibit transcription is a function of the number of lesions.
  • the DNA probe used as a competitor is a PVUI restriction fragment of pSK of 1084 bp, treated (CP (+)) or not (CP (-)) with cisplatin.
  • the TBP also recognizes DNA damaged by cisplatin, which is demonstrated by the nitrocellulose membrane binding test (see example 3 below) (Fig 3C).
  • EXAMPLE 3 Attachment to standard nitrocellulose membrane (Fig 3A and C). Recombinant TBP is incubated with DNA labeled with phosphorus 32, damaged by treatment either with cisplatin (Fig 3C) or by UV irradiation (Fig 3A). The TBP / DNA interaction is determined by the ability of the recombinant TBP to retain DNA on a nitrocellulose membrane.
  • Figures 3A and 3C illustrate this result by showing the percentage of DNA retained on the nitrocellulose filters as a function of the quantity of TBP for four doses of UV and for an undamaged F879 fragment. Quantification is carried out using a phosphoimage analyzer. 100% represents the total obtained when 1 ⁇ l of each DNA probe is fixed on Whatman paper and simultaneously exposed on a nitrocellulose filter.
  • FIG. 3C differs from FIG. 3A only in that, as a DNA probe, a PVUI restriction fragment of 1084 bp of pSK treated (CP (+)) or not (CP (-)) with the cisplatin.
  • UV irradiation of the 328-labeled F879 DNA fragment at increasing irradiation rates (100, 500, 1000 or 1500 J / m 2 ) has a corresponding increase in the amount of DNA retained on the filters. for a given concentration of TBP.
  • the ability of TBP to retain DNA in proportion to the amount of lesions contained in this DNA suggests a specific affinity of TBP for damaged DNA compared to undamaged DNA. Since the F879 fragment does not contain a TATA sequence capable of competing with the binding of TBP, the binding of TBP to this fragment can be directly attributed to the existence of damage.
  • EXAMPLE 4 Migration on acrylamide gel (Fig 4).
  • the ability of purified TBP to bind damaged DNA has also been observed in tests for variation of electrophoretic mobility using the EMSA technique. For that, we incubates a DNA probe (36 mer, 0.5 ng, 10,000 cpm) labeled with phosphorus 32 not damaged or containing only one cisplatin site (1,3-GpTpG) with recombinant TBP and in this second case, the formation of a complex with reduced electrophoretic mobility (20 ng) compared to the free probe is detected on acrylamide gels. In agreement with the membrane fixation tests, the EMSA technique has demonstrated the formation of a TBP / DNA nucleoprotein complex specific for damaged DNA.
  • a significantly larger amount of the damaged DNA sample is mobilized compared to the amount mobilized when the sample is not damaged (Fig 4A, compare columns 2 and 12).
  • the formation of a damaged TBP / DNA complex is reduced significantly when increasing amounts (respectively 10 and 50 ng) of a 64-mer AdMLP fragment containing a TATA sequence are added (columns 5 and 6) or of a fragment of Pvul pSK DNA damaged by cisplatin (respectively 10 and 50 ng) (columns 7 and 8) unlabeled relative to an undamaged fragment (compare with columns 9 and 10).
  • TBP binding is a function of the number of lesions present in DNA and appears to be strong enough to persist in the presence of significant amounts of the TATA sequence.
  • Inhibition of transcription and preferential retention of TBP on a nitrocellulose membrane have also been observed with DNA damaged by acetylaminofluorine (AFF), an aromatic amine known for its carcinogenic capacities.
  • AFF acetylaminofluorine
  • results of the present invention demonstrate the preferential affinity of TBP for damaged DNA regions exhibiting curvature.
  • the method of screening for cytotoxic products can thus be used to discriminate more particularly the products which induce a curvature of the DNA. Such a classification is useful in order to determine the toxic potential and the mode of action of many cytotoxic molecules.
  • DNA repair m an active gene removal of pyrimidme dimers from the DHFR gene of CHO cells is much more efficient than m tne genome overall. Cell voi 40: 359-369.
  • TFIID Yeast TATA-binding protein binds to TATA elements with both consensus and nonconsensus DNA sequences. Proc. Natl. Acad. Sci USA 86: 5718-5722.
  • DNA repair helicase a component of BTF2 (TFIIH) basic transcription factor. Science 260: 58-63.

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Abstract

The invention concerns a method for the isolation and/or assay of damaged DNA in a sample, characterised in that it consists in assessing the importance of the fixation of TBP or of an analogous product on the DNA sample and in determining the results directly or relative to a control. This method can be used for screening cytotoxic products acting on DNA.

Description

PROCEDE DE MISE EN EVIDENCE ET/OU DE DOSAGE D'ADN ENDOMMAGE DANS UN ECHANTILLON D'ADN ET SON APPLICATION AU CRIBLAGE DE PRODUITS CYTOTOXIQUES AGISSANT SUR L'ADNPROCESS FOR EVIDENCE AND / OR DETERMINATION OF DAMAGED DNA IN A DNA SAMPLE AND ITS APPLICATION TO THE SCREENING OF CYTOTOXIC PRODUCTS ACTING ON DNA
La présente invention concerne un procédé de mise en évidence et/ou dosage de la présence d'ADN endommagé dans un échantillon d'ADN et son application à la mise en évidence et/ou au criblage de produits susceptibles d'endommager l'ADN.The present invention relates to a method for detecting and / or assaying the presence of damaged DNA in a DNA sample and its application to the detection and / or screening of products liable to damage DNA.
Les agressions d'origine extérieure que subit l'ADN peuvent être d'origine variée. Elles sont en général regroupées en deux catégories : les agressions d' origine physique produites notamment par les rayonnements, et ; les agressions d'origine chimique.The external aggressions that DNA undergoes can be of various origins. They are generally grouped into two categories: physical aggressions produced in particular by radiation, and; chemical attacks.
Il convient dès à présent de rappeler que l' endommagement de l'ADN est un phénomène qui intervient notamment dans les traitements anticancéreux.It should now be recalled that DNA damage is a phenomenon which occurs in particular in cancer treatments.
Ainsi par exemple, le médicament anticancéreux cisplatine étant considéré comme faisant partie des agents endommageant l'ADN (Zambie et Lippard, 1995), son utilisation dans les traitements de certains cancers est essentielle. C'est pourquoi dans ce qui va suivre, l'évaluation de l' endommagement de l'ADN pourra aussi bien permettre de mettre en évidence la toxicité d'un agent d'agression que de suivre l'évolution d'une thérapie, anticancéreuse notamment. La réponse apportée par les cellules à ces agressions d'origines diverses qui conduisent à l' endommagement de l'ADN, se traduit par la mise en place du mécanisme dit de réparation de l'ADN.For example, since the anticancer drug cisplatin is considered to be one of the DNA damaging agents (Zambia and Lippard, 1995), its use in the treatment of certain cancers is essential. This is why, in what follows, the evaluation of DNA damage could make it possible to highlight the toxicity of an aggressor agent as well as to follow the progress of an anti-cancer therapy. especially. The response provided by cells to these attacks of various origins which lead to DNA damage, results in the establishment of the so-called DNA repair mechanism.
Ce mécanisme de réparation très complexe met en oeuvre un certain nombre de protéines, certaines d'entre elles étant également impliquées dans des processus cellulaires autres que la réparation.This very complex repair mechanism implements a number of proteins, some of which are also involved in cellular processes other than repair.
Ainsi, la possibilité d'un lien entre les mécanismes de transcription de gènes codant pour les protéines et ceux impliqués dans la réparation de l'ADN a été évoquée (Friedberg, 1996) . Plus précisément, le facteur de transcription TFIIH est considéré comme étant essentiel à la fois pour la transcription et pour le mécanisme de réparation des nucleotides par excision (Svejstrup et al;, 1996, Hoeijmakers et al., 1996).Thus, the possibility of a link between the transcription mechanisms of genes coding for proteins and those involved in DNA repair has been discussed (Friedberg, 1996). More specifically, the transcription factor TFIIH is considered to be essential both for transcription and for the mechanism of nucleotide repair by excision (Svejstrup et al;, 1996, Hoeijmakers et al., 1996).
Toutefois l' importance, dans le mécanisme de reconnaissance d'une lésion à l'ADN, d'une protéine connue pour son activité au niveau de la transcription, dénommée TBP pour "TATA binding protein", n'avait jamais été suggérée et c'est précisément cette constatation qui a permis de développer les procédés de dosage, objet de la présente invention.However, the importance, in the mechanism of recognition of a DNA lesion, of a protein known for its activity at the level of transcription, called TBP for "TATA binding protein", had never been suggested and c is precisely this observation which has made it possible to develop the assay methods which are the subject of the present invention.
En effet, on a pu mettre en évidence qu'en cas d' endommagement de l'ADN, d'une part le TBP se lie à l'ADN endommagé pour former un complexe qui peut être identifié et dosé, et d'autre part que la fixation de ce TBP sur l'ADN endommagé constitue un facteur limitant pour la transcription d'un ADN non endommagé. Ainsi, pour évaluer l' endommagement de l'ADN, on peut mettre à profit la fixation du TBP sur l'ADN endommagé et par la même le déficit de TBP dans le milieu, par la diminution de la transcription d'un ADN non endommagé.Indeed, it has been possible to demonstrate that in the event of DNA damage, on the one hand the TBP binds to the damaged DNA to form a complex which can be identified and assayed, and on the other hand that the binding of this TBP to damaged DNA constitutes a limiting factor for the transcription of an undamaged DNA. Thus, to evaluate the DNA damage, one can take advantage of the fixation of TBP on the damaged DNA and by the same the deficit of TBP in the medium, by the reduction of the transcription of an undamaged DNA. .
C'est pourquoi, la présente invention concerne un procédé de mise en évidence et/ou de dosage d'ADN endommagé dans un échantillon, caractérisé en ce qu'on évalue l'importance de la fixation du TBP ou d'un produit analogue sur l'ADN de l'échantillon et on détermine les résultats directement ou par rapport à un témoin.This is why, the present invention relates to a method for detecting and / or assaying damaged DNA in a sample, characterized in that the importance of the fixation of TBP or of a similar product is evaluated on the DNA of the sample and the results are determined directly or relative to a control.
Par TBP, on entend aussi bien le TBP sous forme 'libre, par exemple une protéine purifiée recombinante que sous sa forme complexée, par exemple le complexe TBP/THIID. D'une manière générale, on peut aussi envisager d'utiliser un produit analogue au TBP qui conserve sa capacité à fixer l'ADN endommagé.TBP is understood to mean both TBP in free form, for example a purified recombinant protein and in its complexed form, for example the TBP / THIID complex. In general, one can also consider using a product similar to TBP which retains its ability to fix damaged DNA.
Par dosage, on entend aussi bien un dosage qualitatif que quantitatif. Par évaluation, on entend déterminer la fixation ou la non fixation du TBP, laquelle est faite directement si on dispose par exemple de courbes d'étalonnage, ou bien par comparaison avec un témoin correspondant à des ADN non endommagés ou ADN endommagés témoins. Comme cela sera démontré ci-après, le complexe ADN endommagé/TBP est suffisamment stable pour être identifié et dosé et l'importance de la fixation de TBP est un bon indice de l'importance du dommage causé à l'ADN.By dosage is meant both a qualitative and a quantitative dosage. The term “evaluation” is intended to determine the fixation or non-fixation of the TBP, which is done directly if, for example, calibration curves are available, or else by comparison with a control corresponding to undamaged DNA or damaged DNA controls. As will be demonstrated below, the damaged DNA / TBP complex is stable enough to be identified and assayed and the extent of TBP binding is a good indication of the extent of damage to DNA.
Par importance, on entend désigner aussi bien la qualité ou le type de dommages que leur nombre, voire même l' emplacement de ces dommages .By importance is meant both the quality or type of damage as well as the number and even the location of the damage.
La mise en évidence ou le dosage peuvent être effectués soit directement soit indirectement.The demonstration or the assay can be carried out either directly or indirectly.
Dans le premier cas, on utilise comme élément à doser le complexe formé lui-même, dans le second cas, on utilise le fait que lorsqu'elle est engagée dans le complexe TBP/ADN endommagé, le TBP n'est plus disponible pour assurer l'initiation de la transcription. Il y a donc une diminution de la transcription qui est proportionnelle au TBP fixé sur l'ADN endommagé et au dommage de l'ADN.In the first case, the complex formed itself is used as the dosing element, in the second case, the fact that when it is engaged in the damaged TBP / DNA complex, the TBP is no longer available to ensure initiation of transcription. There is therefore a decrease in transcription which is proportional to the TBP attached to the damaged DNA and to the damage to the DNA.
Ainsi, dans un premier mode de réalisation, la présente invention concerne un procédé de mise en évidence et/ou de dosage d'ADN endommagé dans un échantillon caractérisé en ce que l'importance de la fixation du TBP sur l'ADN est évaluée par détermination de la transcription d'un ADN non endommagé connu, dans un milieu assurant la transcription, la transcription n'étant assurée que par la présence dans ledit milieu de TBP résiduel non fixé sur l'ADN.Thus, in a first embodiment, the present invention relates to a method for detecting and / or assaying damaged DNA in a sample characterized in that the importance of the binding of TBP to the DNA is evaluated by determination of the transcription of a known undamaged DNA, in a medium ensuring transcription, transcription being ensured only by the presence in said medium of residual TBP not fixed on DNA.
Comme cela a été précisé précédemment, le TBP étant fixé à l'ADN endommagé, il n'est pas disponible pour assurer la transcription de l'ADN non endommagé.As previously stated, since TBP is attached to damaged DNA, it is not available to transcribe undamaged DNA.
La mesure de la transcription, et donc de l'ARN transcrit correspondant à l'ADN non endommagé, peut être effectuée par toutes méthodes connues, migration sur gel par exemple, comme cela sera expliqué plus en détails dans les exemples ci-après.The measurement of the transcription, and therefore of the transcribed RNA corresponding to the undamaged DNA, can be carried out by all known methods, migration on gel for example, as will be explained in more detail in the examples below.
De préférence, le procédé est mis en oeuvre de la façon suivante : a) l'échantillon est mis en incubation avec du TBP ou un produit analogue ; b) après incubation, on ajoute à l'échantillon une séquence d'ADN non endommagé connu, dans des conditions assurant la transcription mais sans TBP additionnel ; c) on détermine la transcription de cet ADN connu et ; d) on mesure la variation de transcription de cet ADN connu par rapport à une transcription normale ou par comparaison avec un témoin.Preferably, the method is carried out as follows: a) the sample is incubated with TBP or a similar product; b) after incubation, a known undamaged DNA sequence is added to the sample, under conditions ensuring transcription but without additional TBP; c) determining the transcription of this known DNA and; d) the variation in transcription of this known DNA is measured with respect to a normal transcription or by comparison with a control.
De façon générale, l'étape a) est effectuée dans des conditions qui ne permettent pas la réparation de l'ADN et l'étape b) est effectuée dans un milieu qui peut être constitué d' extraits cellulaires totaux, extraits de cellules HeLa par exemple ou encore d'un système de transcription reconstitué.In general, step a) is carried out under conditions which do not allow DNA repair and step b) is carried out in a medium which can consist of total cellular extracts, extracted from HeLa cells by example or of a reconstructed transcription system.
Dans un second mode de réalisation du procédé selon l'invention, on met en évidence et/ou on dose la présence d'ADN endommagé dans un échantillon par un procédé dans lequel ledit échantillon est mis en présense d'un réactif contenant du TBP ou un produit analogue et on évalue l'importance du dommage de l'ADN par mise en évidence et/ou dosage du complexe T3P/ADN formé, directement ou par rapport à un témoin.In a second embodiment of the method according to the invention, the presence of damaged DNA is detected and / or measured in a sample by a method in which said sample is placed in the presence of a reagent containing TBP or an analogous product and the extent of the DNA damage is evaluated by detecting and / or assaying the T3P / DNA complex formed, directly or in relation to a control.
Le complexe TBP/ADN peut être mis en évidence et/ou dosé par toute méthode appropriée.The TBP / DNA complex can be demonstrated and / or determined by any suitable method.
On peut tout d'abord évaluer et doser le complexe lui-même, par exemple en le fixant sur un support présentant une affinité pour les protéines. Il peut s'agir notamment d'une membrane de nitrocellulose et on mesurera alors la quantité d'ADN retenue sur la membrane.We can first of all evaluate and measure the complex itself, for example by fixing it on a support having an affinity for proteins. It may especially be a nitrocellulose membrane and the quantity of DNA retained on the membrane will then be measured.
On peut également mettre à profit les propriétés particulières du complexe formé. Par exemple, on peut mesurer la variation de mobilité du complexe sur un gel, gel d' acrylamide par exemple.One can also take advantage of the particular properties of the complex formed. For example, we can measure the variation of mobility of the complex on a gel, for example acrylamide gel.
Enfin, on peut envisager d'évaluer la formation du complexe par la technique d'empreinte à la Dnase I. On peut bien entendu effectuer un dosage du TBP libre restant, lorsque l'on connait la quantité de TBP introduite à l'origine, le TBP libre peut être dosé par toute méthode connue pour doser les protéines, méthode ELISA par exemple.Finally, it is possible to envisage evaluating the formation of the complex by the Dnase I imprint technique. It is of course possible to carry out an assay of the remaining free TBP, when we know the quantity of TBP originally introduced the free TBP can be assayed by any known method for assaying proteins, ELISA method for example.
L' étude du complexe formé TBP/ADN endommagé a conduit de façon surprenante à mettre en évidence une structure de l'ADN analogue à celle résultant de la reconnaissance de la séquence TATA par le TBP.The study of the complex formed TBP / damaged DNA led, surprisingly, to reveal a DNA structure analogous to that resulting from the recognition of the TATA sequence by TBP.
Sous l'action des lésions, l'ADN adopte une structure courbée qui présente des similitudes avec la forme A de l'ADN. L'élargissement et l'aplatissement du petit sillon qui en résulte, permettent des contacts hydrophobes et favorisent la formation du complexe TBP/ADN endommagé.Under the action of lesions, DNA adopts a curved structure which presents similarities to form A of DNA. The resulting enlargement and flattening of the small groove allows hydrophobic contacts and promotes the formation of the damaged TBP / DNA complex.
L'analogie a ainsi permis de démontrer que des fragments d'ADN de "TATA box" étaient susceptibles d'entrer en compétition avec l'ADN endommagé lors de la formation du complexe.The analogy thus made it possible to demonstrate that DNA fragments from the "TATA box" were capable of competing with the DNA damaged during the formation of the complex.
Il est ainsi possible de prévoir des dosages par compétition.It is thus possible to provide dosages per competition.
Les procédés selon la présente invention permettent de mettre non seulement en évidence l' endommagement de l'ADN marqué, mais également son dosage semi-quantitatif ou quantitatif.The methods according to the present invention make it possible to highlight not only the damage to the labeled DNA, but also its semi-quantitative or quantitative assay.
Notamment l'utilisation de réactifs radioactifs ou non, par exemple fluorescents, permet par mesure d'une intensité de bande de faire un dosage quantitatif. L'intérêt de ces dosages est tout d'abord, au stade de la recherche industrielle, de permettre de la recherche de produits cytotoxiques ayant un mode d'action particulier de type "cisplatine" . Ainsi, le procédé selon la présente invention peut être utilisé comme un procédé de criblage de produits cytotoxiques sur l'ADN, notamment les anticancéreux, ceux-ci étant révélés soit par l'apparition du complexe TBP/ADN, soit par la diminution de la transcription d'un ADN qui n'a pas été endommagé.In particular, the use of radioactive or non-radioactive reagents, for example fluorescent, makes it possible by measuring a band intensity to make a quantitative assay. The interest of these assays is first of all, at the stage of industrial research, to allow the search for cytotoxic products having a particular mode of action of the "cisplatin" type. Thus, the method according to the present invention can be used as a method for screening for cytotoxic products on DNA, in particular anticancer drugs, these being revealed either by the appearance of the TBP / DNA complex, or by the decrease in the transcription of DNA that has not been damaged.
Les procédés selon la présente invention peuvent également être utilisés pour tester la toxicité éventuelle de certains produits, cosmétiques, herbicides, pharmaceutiques, par exemple par des prélèvements réguliers dans des enceintes industrielles à risque.The methods according to the present invention can also be used to test the possible toxicity of certain products, cosmetics, herbicides, pharmaceuticals, for example by regular sampling in industrial enclosures at risk.
Le procédé selon la présente invention peut également être utilisé pour le suivi thérapeutique, notamment dans les thérapies anticancéreuses. Plus précisément, pour déterminer la cytotoxicité sur l'ADN d'un produit donné, on fait réagir au préalable ledit produit dans un échantillon d'ADN non endommagé, ledit échantillon étant alors dépourvu de tous autres produits susceptibles d' endommager l'ADN et on procède selon les différentes variantes envisagées selon l' invention, la cytotoxicité du produit étant établie lorsque de l'ADN endommagé est détecté.The method according to the present invention can also be used for therapeutic monitoring, in particular in anticancer therapies. More specifically, to determine the cytotoxicity on the DNA of a given product, said product is reacted beforehand in an undamaged DNA sample, said sample then being free of any other products capable of damaging DNA and one proceeds according to the different variants envisaged according to the invention, the cytotoxicity of the product being established when damaged DNA is detected.
La présente invention sera illustrée par les exemples suivants accompagnées des figures 1 à 7, lesquels représentent : Figure 1 : Partie (A) : Schématise une expérience de compétition / transcription.The present invention will be illustrated by the following examples accompanied by FIGS. 1 to 7, which represent: FIG. 1: Part (A): Schematizes a competition / transcription experience.
Partie (B) : Schématise la structure de différents fragments d'ADN utilisés pour l'expérience de compétition / transcription in vitro. Une matrice de transcription AdMLP est générée par digestion/restriction avec EcoRI/SalI du plasmide pUC309. Elle donne naissance à un transcrit d'ARN de 309 nucleotides qui contient le promoteur AdMLP (partie hachurée). Le fragment F879 est créé par digestion/restriction de pUC309 avec BamHI et Sspl . Ce fragment ne contient aucune séquence promotrice .Part (B): Schematizes the structure of different DNA fragments used for the in vitro competition / transcription experiment. An AdMLP transcription matrix is generated by digestion / restriction with EcoRI / SalI of the plasmid pUC309. It gives rise to an RNA transcript of 309 nucleotides which contains the promoter AdMLP (hatched part). The F879 fragment is created by digestion / restriction of pUC309 with BamHI and Sspl. This fragment does not contain any promoter sequence.
Figure 2 (A et B) : Représente les résultats obtenus lors d'expériences d'inhibition de la transcription in vitro à partir du gène AdMLP en présence d'ADN endommagé par les UV ou par le cisplatine.Figure 2 (A and B): Shows the results obtained in experiments of inhibition of transcription in vitro from the AdMLP gene in the presence of DNA damaged by UV rays or by cisplatin.
Figure 3 (A à D) : Représente des dosages réalisés en utilisant la fixation du complexe sur une membrane de cellulose. Figure 4 (A et B) : Représente les résultats des dosages réalisés en utilisant la fixation du complexe sur une membrane de nitrocellulose en présence de fragments compétiteurs.Figure 3 (A to D): Represents assays performed using the fixation of the complex on a cellulose membrane. Figure 4 (A and B): Shows the results of the assays performed using the fixation of the complex on a nitrocellulose membrane in the presence of competing fragments.
Les expériences suivantes ont été réalisées pour mettre en évidence la capacité du TBP à interagir avec de l'ADN endommagé. MATERIELS ET METHODES On utilise des extraits cellulaires totaux ( CΞ) HeLa ainsi que les composants du système de transcription reconstitué in vitro tel que décrit dans Humbert et al. (1994).The following experiments were performed to demonstrate the ability of TBP to interact with damaged DNA. MATERIALS AND METHODS Total cellular extracts (CΞ) HeLa are used as well as the components of the transcription system reconstituted in vitro as described in Humbert et al. (1994).
Les substrats utilisés pour la transcription in vitro ou pour le test de fixation sur membrane sont préparés par digestion/restriction soit d'ADN plasmidique de pUC309 soit de pSK. Le plasmide pUC309 est préparé par ligation d'un fragmentThe substrates used for in vitro transcription or for the membrane binding test are prepared by digestion / restriction either of plasmid DNA from pUC309 or from pSK. Plasmid pUC309 is prepared by ligation of a fragment
EcoRI/BamHI correspondant aux séquences -372 à +33 de AdMLP, au fragment BamHI/SalI de pBR322. Le fragment résultant est clone dans les sites EcoRI/SalI de pUC19 pour former le plasmide pUC309. Le fragment d'ADN compétiteur de 879 pb (F879) est préparé par digestion/restriction de pUC309 avec BamHI/SspIEcoRI / BamHI corresponding to the sequences -372 to +33 of AdMLP, to the BamHI / SalI fragment of pBR322. The resulting fragment is cloned into the EcoRI / SalI sites of pUC19 to form the plasmid pUC309. The competing DNA fragment of 879 bp (F879) is prepared by digestion / restriction of pUC309 with BamHI / SspI
(Figure 1B) . Les fragments linéaires sont purifiés sur gel d'agarose à 1% et billes Qiaex (Société Qiagen) . L'ADN est resuspendu dans un tampon Tris-HCl 50 mM (pH 7.9), contenant 10 % de glycerol, ImM d'EDTA, 0,5 mM de DTT et 50 mM de KCl pour utilisation dans les expériences de transcription ou de mise en évidence de complexe Protéine/ADN (EMSA ; rétention sur membrane) . Le compétiteur final utilisé était le "Bluescript" de(Figure 1B). The linear fragments are purified on 1% agarose gel and Qiaex beads (Qiagen company). The DNA is resuspended in a 50 mM Tris-HCl buffer (pH 7.9), containing 10% glycerol, ImM of EDTA, 0.5 mM of DTT and 50 mM of KCl for use in the transcription or setting experiments. evidence of protein / DNA complex (EMSA; membrane retention). The final competitor used was the "Bluescript" of
3 kb, plasmide pSK (Stratagene) . Le fragment F879 a été endommagé aux UV avec une lampe UV-C à une puissance 0.1 m /cm2. Le plasmide pSK est traité avec une solution de 0.1 mg/ml dans l'eau de dichlorure de cisplatine ( II ) diamine (Sigma) pendant 15 heures dans l'obscurité à 37°C et avec un rapport cisplatine/nucléotide de 0.005 (Hansson and ood, 1989).3 kb, plasmid pSK (Stratagene). The F879 fragment was UV-damaged with a UV-C lamp at a power of 0.1 m / cm 2 . The plasmid pSK is treated with a solution of 0.1 mg / ml in water of cisplatin (II) dichloride diamine (Sigma) for 15 hours in the dark at 37 ° C. and with a cisplatin / nucleotide ratio of 0.005 (Hansson and ood, 1989).
On génère les fragments CP(-) ou CP(+) utilisés ultérieurement dans les tests de fixation sur membrane par digestion/restriction de pSK, respectivement intacts ou endommagés par le cisplatine, avec l'enzyme de restriction Pvul suivie d'une purification du fragment de 1084 pb sur des gels d' agarose 1% sans BET .The CP (-) or CP (+) fragments are used which are used subsequently in the membrane binding tests by digestion / restriction of pSK, respectively intact or damaged by cisplatin, with the restriction enzyme Pvul followed by purification of the 1084 bp fragment on 1% agarose gels without BET.
Pour la technique EMSA, un ADN compétiteur (AdMLP (64 mer)) a été préparé à partir d' oligonucléotides complémentaires synthétisés correspondant aux régions -40 à +24 du promoteur majeur tardif de l'adénovirus 2 (AdMLP).For the EMSA technique, a competitive DNA (AdMLP (64 mer)) was prepared from complementary synthesized oligonucleotides corresponding to the regions -40 to +24 of the late major promoter of adenovirus 2 (AdMLP).
On fait réagir la sonde 32-mer 5' -TCTTCTTCTTCTTCTGTGCACTCTTCTTCTCT-3' contenant un seul GpTpG avec du cisplatine (Moggs et al., 1996). Après précipitation à l'éthanol, la présence d'une liaison 1, 3-cisplatine d(GpTpG) a été confirmée par analyse de l' oligonucleotide sur un gel d' acrylamide à 12 %, l' oligonucleotide modifié migrant plus lentement que l' oligonucleotide intact.The 32-mer 5 '-TCTTCTTCTTCTTCTGTGCACTCTTCTTCTCT-3' probe containing a single GpTpG is reacted with cisplatin (Moggs et al., 1996). After ethanol precipitation, the presence of a 1,3-cisplatin d (GpTpG) bond was confirmed by analysis of the oligonucleotide on a 12% acrylamide gel, the modified oligonucleotide migrating more slowly than l intact oligonucleotide.
La sonde d'ADN résultante (36 pb) utilisée dans le test EMSA est préparée en appariant l' oligonucleotide cisplatine avec son oligonucleotide complémentaire non endommagé laissant une partie 5' libre à chaque extrémité.The resulting DNA probe (36 bp) used in the EMSA test is prepared by pairing the oligonucleotide cisplatin with its complementary undamaged oligonucleotide leaving a free 5 'portion at each end.
L'ADN est complété avec incorporation de P32 dATP, marqué radioactivement avec P32 dATP (3000-Ci/mmol) en présence du fragment de Klenow de l'ADN polymérase I d'E.coli puis purifié sur colonnes sephadex G50.The DNA is completed with incorporation of P32 dATP, radioactively labeled with P32 dATP (3000-Ci / mmol) in the presence of the Klenow fragment of the DNA polymerase I of E. coli and then purified on sephadex G50 columns.
Pour la technique EMSA, on prépare des milieux de réaction (20-μl) contenant un tampon 50 mM Tris/HCl pH 7.9, 0.2 ng d'une sonde d'ADN de 36 pb marquée au P32 (10000 cpm) 80 mM de KCl, 5 mM de MgCl2, 0.1 mM de EDTA, 500 ng de BSA, 10- de glycerol, 0,5 mM de DTT, 0.01 % NP40, TBP recombinant. Après 30-minutes d'incubation à 25°C, les échantillons sont ajustés à 20 % de glycerol et déposés sur gel de polyacrylamide à 4 %. La migration électrophorétique se fait dans un tampon 25m MTris/19mM Glycine à température ambiante. Les gels sont séchés et exposés aux films biomax (Kodak) .For the EMSA technique, reaction media (20 μl) are prepared containing a 50 mM Tris / HCl buffer pH 7.9, 0.2 ng of a 36 bp DNA probe marked with P32 (10,000 cpm) 80 mM KCl , 5 mM of MgCl2, 0.1 mM of EDTA, 500 ng of BSA, 10- of glycerol, 0.5 mM DTT, 0.01% NP40, recombinant TBP. After 30 minutes of incubation at 25 ° C, the samples are adjusted to 20% glycerol and deposited on 4% polyacrylamide gel. Electrophoretic migration takes place in a 25m MTris / 19mM Glycine buffer at room temperature. The gels are dried and exposed to biomax films (Kodak).
EXEMPLE 1 : Procédé utilisant le "défaut de transcription" du à la formation du complexe TBP/ADN (Fig. 2).EXAMPLE 1: Method using the "transcription defect" due to the formation of the TBP / DNA complex (FIG. 2).
On incube 20-30 μg d'extraits cellulaires totaux préparés à partir de cellules HeLa avec des quantités croissantes d'ADN endommagé (matrice compétiteur) dans un tampon 50 mM Tris/HCl pH 7.9 contenant 10 % de glycerol, ImM de EDTA, 0,5 mM de DTT, et 5 mM de MgC12 pendant 15 min à 25°C (volume final des produits réactionnels = 20 μl) afin de permettre la fixation de TBP/TFIID.Incubate 20-30 μg of total cell extracts prepared from HeLa cells with increasing amounts of damaged DNA (competitive matrix) in a 50 mM Tris / HCl pH 7.9 buffer containing 10% glycerol, ImM of EDTA, 0 , 5 mM DTT, and 5 mM MgC12 for 15 min at 25 ° C (final volume of the reaction products = 20 μl) in order to allow the fixation of TBP / TFIID.
On ajoute ensuite 90 ng de la matrice AdMLP (EcoRI/SalI) devant donner naissance à un transcript spécifique que l'on laisse incuber pendant 15 minutes pour favoriser la formation du complexe de préinitiation de la transcription. Suite à l'addition de nucleotides triphosphate ATP, GTP, UTP, y compris p32 CTP (400Ci/mmol) , (volume final de réaction de 25 μl, la transcription demeurera et se poursuivra pendant 45 min à 25 °C. La réaction est stoppée par addition d'une solution contenant du sodium docecyl sulfate à 10 % (p.V.), les ARN transcrits sont analysés après électrophorèse sur gel 5% acrylamide, par autoradiographie puis quantifiés directement par comptage sur un Analyseur Phospholmage ou indirectement par balayage densitométrique des autoradiogrammes en utilisant un densitomètre à image BioRad GS700. Les résultats de ce dosage sont représentés Figure 2.90 ng of the AdMLP matrix (EcoRI / SalI) are then added which should give rise to a specific transcript which is left to incubate for 15 minutes to promote the formation of the transcription pre-initiation complex. Following the addition of nucleotides triphosphate ATP, GTP, UTP, including p32 CTP (400Ci / mmol), (final reaction volume of 25 μl, transcription will remain and will continue for 45 min at 25 ° C. The reaction is stopped by the addition of a solution containing sodium docecyl sulfate at 10% (pV), the transcribed RNAs are analyzed after electrophoresis on 5% acrylamide gel, by autoradiography and then quantified directly by counting on a phospholmage analyzer or indirectly by densitometric scanning of autoradiograms using a BioRad GS700 image densitometer The results of this assay are shown in Figure 2.
Partie (A) : Les résultats de la transcription de l' AdMLP réalisée avec 30 μg de WCE en présence de quantités croissantes du fragment d'ADN F879 irradié aux UV (650 J/m2) (colonnes 3-8) ou non irradiés (colonnes 9-14). La partie supérieure de la figure représente un autoradiogramme, la partie inférieure représente la quantification densitométrique de 1' autoradiogramme (bande de 309 nucleotides) : le pourcentage de transcription à partir de AdMLP est exprimé en fonction de la quantité d'ADN F879 compétiteur exprimée en ng .Part (A): The results of the transcription of AdMLP carried out with 30 μg of WCE in the presence of increasing amounts of DNA fragment F879 irradiated with UV (650 J / m2) (columns 3-8) or not irradiated ( columns 9-14). The upper part of the figure represents an autoradiogram, the lower part represents the densitometric quantification of the autoradiogram (band of 309 nucleotides): the percentage of transcription from AdMLP is expressed as a function of the quantity of competing F879 DNA expressed in ng.
Partie (B) : Les résultats de la transcription de AdMLP réalisée avec 20 μg de CE en présence de quantités croissantes de pSK non endommagés (CP(-)) (colonnes 2-8) ou endommagées par le cisplatine (CP(+)) (colonnes 9-15). La partie supérieure de la figure représente la production de transcripts à partir de l'AdMLP (bande de 309 nucleotides). La partie inférieure de la figure représente le même type de quantification que la partie (A) .Part (B): The results of the transcription of AdMLP carried out with 20 μg of EC in the presence of increasing amounts of undamaged pSK (CP (-)) (columns 2-8) or damaged by cisplatin (CP (+)) (columns 9-15). The upper part of the figure represents the production of transcripts from AdMLP (band of 309 nucleotides). The lower part of the figure represents the same type of quantification as part (A).
Cet exemple montre clairement le piégeage par l'ADN endommagé aux UV (UV(+ )) ou au cisplatine (CP( +)) ou l'AAF de facteurs, qui de ce fait ne seront plus disponibles pour la réaction de transcription. La synthèse de transcripts diminue proportionnellement à la quantité d'ADN endommagé ajoutée. On constate également qu' il est possible de quantifier grâce à la transcription la nature et le nombre des lésions.This example clearly shows trapping by DNA damaged by UV (UV (+)) or cisplatin (CP (+)) or AAF factors, which therefore will no longer be available for the transcription reaction. The synthesis of transcripts decreases in proportion to the amount of damaged DNA added. It is also noted that it is possible to quantify by means of the transcription the nature and the number of lesions.
La pré-incubation des extraits cellulaires totaux (WCΞ) avec le fragment de 879 paires de bases irradié aux UV F879 UV(+) (Figure 1B) conduit à une inhibition de la transcription de la matrice AdMLP qui est quatre fois supérieure à celle observée lorsque l'ADN est intact (Figure 2A, comparer les colonnes 3-8 avec les colonnes 9-14 et voir la partie inférieure de la figure) . De la même façon, la pré-incubation du plasmide pSK de 3kb contenant environ 30 sites cisplatines (Hannon et al. 1989) inhibe la transcription environ trois fois plus qu'en présence d'ADN intact (Figure 2B, comparer les colonnes 2-8 aux colonnes 9-15 et voir la partie inférieure de la figure) .Pre-incubation of total cell extracts (WCΞ) with the fragment of 879 base pairs irradiated with UV F879 UV (+) (Figure 1B) leads to an inhibition of the transcription of the AdMLP matrix which is four times greater than that observed when the DNA is intact (Figure 2A, compare columns 3-8 with columns 9-14 and see the lower part of the figure). Similarly, pre-incubation of the 3kb pSK plasmid containing approximately 30 cisplatin sites (Hannon et al. 1989) inhibits transcription approximately three times more than in the presence of intact DNA (Figure 2B, compare columns 2- 8 in columns 9-15 and see the lower part of the figure).
Afin de confirmer que l'inhibition de la transcription du transcrit de 309 nucleotides était bien liée à l'effet de l'AD endommagé sur des facteurs nécessaires à la transcription, une concentration fixe d'ADN compétiteur a été ajoutée dans le milieu réactionnel tout en faisant varier le rapport fragment endommagé/fragment intact. Dans ces conditions, on a observé une inhibition de la transcription qui augmente avec la quantité de fragments d'ADN endommagé présents dans le milieu réactionnel (Figure 2A, colonnes 15-20) .In order to confirm that the inhibition of transcription of the 309 nucleotide transcript was indeed linked to the effect of damaged AD on factors necessary for transcription, a fixed concentration of competitor DNA was added to the reaction medium while varying the damaged fragment / intact fragment ratio. Under these conditions, an inhibition of transcription was observed which increases with the quantity of damaged DNA fragments present in the reaction medium (FIG. 2A, columns 15-20).
EXEMPLE 2 : Diminution de la transcription dans un système de transcription reconstitué (RTS) (Fig. 3B et D) .EXAMPLE 2: Decrease in transcription in a reconstituted transcription system (RTS) (Fig. 3B and D).
Dans cet exemple on utilise un système de transcription reconstitué hautement purifié (RTS), qui contient les facteurs de transcription, TFIIA, TFIIB, le complexe TFIID/TBP, TFIIE, TFIIF et l'ARN pol II. Ce système qui contient tous les facteurs essentiels à la transcription, est pré-incubé avec l'ADN F879 endommagé par irradiation avec 500 (col. 6 à 9) ou 1000 (col. 10 à 13) J/m2 et testé pour sa capacité à inhiber la transcription à partir d'une matrice du gène reporteur AdMLP. La présence d'ADN endommagé inhibe de façon préférentielle la production du transcrit de 309 nucleotides (Fig-3B), ce qui est en accord avec les observations précédentes et la fixation sur membrane de nitrocellulose . On observe une inhibition proportionnelle à la quantitié d'ADN endommagé et donc à la quantité des lésions dues aux UV (comparer les colonnes 2-5 avec les colonnes 6-9 et 10- 13) ou dues au cisplatine (Fig 3D) (comparaison des colonnes 2 et 3 avec les colonnes 4 et 5), ce qui démontre que la capacité de l'ADN endommagé à inhiber la transcription est fonction du nombre de lésions. Dans cet exemple, la sonde d'ADN utilisée en tant que compétiteur est un fragment de restriction PVUI de pSK de 1084 pb, traité (CP(+)) ou non (CP(-)) au cisplatine. Le TBP reconnaît également de l'ADN endommagé par le cisplatine, ce qui est démontré par le test de fixation sur membrane de nitrocellulose (voir exemple 3 ci-après) (Fig 3C) . Il est possible de rétablir complètement la transcription après ajout supplémentaire de TBP (Fig 3D, colonnes 6 et 7). La stimulation de la transcription ainsi observée est très largement supérieure à celle observée en l'absence d'ADN endommagé (colonnes 8 et 9) . EXEMPLE 3 : Fixation sur membrane de nitrocellulose standard (Fig 3A et C) . On met à incuber du TBP recombinant avec de l'ADN marqué au phosphore 32, endommagé par traitement soit par le cisplatine (Fig 3C) , soit par irradiation UV (Fig 3A) . L'interaction TBP/ADN est déterminée par la capacité du TBP recombinant à retenir l'ADN sur une membrane de nitrocellulose. Les Figures 3A et 3C illustrent ce résultat en montrant le pourcentage d'ADN retenu sur les filtres de nitrocellulose en fonction de la quantité de TBP pour quatre doses d'UV et pour un fragment F879 non endommagé. La quantification est réalisée en utilisant un analyseur phosphoimage . 100 % représente le total obtenu lorsque 1 μl de chaque sonde d'ADN est fixé sur un papier Whatman et simultanément exposé sur filtre de nitrocellulose.In this example, a highly purified reconstituted transcription system (RTS) is used, which contains the transcription factors, TFIIA, TFIIB, the TFIID / TBP complex, TFIIE, TFIIF and RNA pol II. This system, which contains all the factors essential for transcription, is pre-incubated with DNA F879 damaged by irradiation with 500 (col. 6 to 9) or 1000 (col. 10 to 13) J / m 2 and tested for its ability to inhibit transcription from a matrix of the reporter gene AdMLP. The presence of damaged DNA preferentially inhibits the production of the 309 nucleotide transcript (FIG. 3B), which is in agreement with the previous observations and the binding to the nitrocellulose membrane. An inhibition is observed proportional to the quantity of damaged DNA and therefore to the amount of lesions due to UV (compare columns 2-5 with columns 6-9 and 10-13) or due to cisplatin (Fig 3D) (comparison columns 2 and 3 with columns 4 and 5), which demonstrates that the capacity of damaged DNA to inhibit transcription is a function of the number of lesions. In this example, the DNA probe used as a competitor is a PVUI restriction fragment of pSK of 1084 bp, treated (CP (+)) or not (CP (-)) with cisplatin. The TBP also recognizes DNA damaged by cisplatin, which is demonstrated by the nitrocellulose membrane binding test (see example 3 below) (Fig 3C). It is possible to completely restore the transcription after additional TBP addition (Fig 3D, columns 6 and 7). The stimulation of the transcription thus observed is very much greater than that observed in the absence of damaged DNA (columns 8 and 9). EXAMPLE 3: Attachment to standard nitrocellulose membrane (Fig 3A and C). Recombinant TBP is incubated with DNA labeled with phosphorus 32, damaged by treatment either with cisplatin (Fig 3C) or by UV irradiation (Fig 3A). The TBP / DNA interaction is determined by the ability of the recombinant TBP to retain DNA on a nitrocellulose membrane. Figures 3A and 3C illustrate this result by showing the percentage of DNA retained on the nitrocellulose filters as a function of the quantity of TBP for four doses of UV and for an undamaged F879 fragment. Quantification is carried out using a phosphoimage analyzer. 100% represents the total obtained when 1 μl of each DNA probe is fixed on Whatman paper and simultaneously exposed on a nitrocellulose filter.
La Figure 3C diffère de la figure 3A seulement en ce que, en tant que sonde d'ADN, on utilise un fragment de restriction PVUI de 1084 pb de pSK traité (CP( +)) ou non (CP(-)) avec le cisplatine. L'irradiation aux UV du fragment d'ADN F879 marqué au 32P à des taux d'irradiation croissants (100, 500, 1000 ou 1500 J/m2) a pour effet une augmentation correspondante de la quantité d'ADN retenue sur les filtres pour une concentration donnée de TBP. La capacité du TBP à retenir l'ADN de façon proportionnelle à la quantité de lésions contenues dans cet ADN suggère une affinité spécifique de TBP pour l'ADN endommagé par rapport à l'ADN non endommagé. Le fragment F879 ne contenant pas de séquence TATA susceptible de concurrencer la liaison du TBP, la fixation de TBP sur ce fragment peut être directement imputée à l'existence d'un dommage.FIG. 3C differs from FIG. 3A only in that, as a DNA probe, a PVUI restriction fragment of 1084 bp of pSK treated (CP (+)) or not (CP (-)) with the cisplatin. UV irradiation of the 328-labeled F879 DNA fragment at increasing irradiation rates (100, 500, 1000 or 1500 J / m 2 ) has a corresponding increase in the amount of DNA retained on the filters. for a given concentration of TBP. The ability of TBP to retain DNA in proportion to the amount of lesions contained in this DNA suggests a specific affinity of TBP for damaged DNA compared to undamaged DNA. Since the F879 fragment does not contain a TATA sequence capable of competing with the binding of TBP, the binding of TBP to this fragment can be directly attributed to the existence of damage.
EXEMPLE 4 : Migration sur gel d' acrylamide (Fig 4). La capacité de TBP purifié à se lier à l'ADN endommagé a été également observée dans des tests de variation de mobilité électrophorétique en utilisant la technique EMSA. Pour cela, on met à incuber une sonde d'ADN (36 mer, 0,5 ng, 10.000 cpm) marquée au phosphore 32 non-endommagé ou ne contenant qu'un seul site cisplatine (1,3-GpTpG) avec du TBP recombinant et dans ce second cas, on détecte la formation d'un complexe ayant une mobilité electrophoretique (20ng) réduite par rapport à la sonde libre sur des gels d' acrylamide . En accord avec les tests de fixation sur membrane, la technique EMSA a mis en évidence la formation d' un complexe nucléoprotéique TBP/ADN spécifique de l'ADN endommagé. En présence de TBP, une quantité nettement plus importante de l'échantillon d'ADN endommagé est mobilisée par rapport à la quantité mobilisée lorsque l'échantillon n'est pas endommagé (Fig 4A, comparer les colonnes 2 et 12). En outre, on constate que la formation d'un complexe TBP/ADN endommagé est réduite de façon significative lorsque l'on ajoute des quantités croissantes (respectivement 10 et 50 ng) d'un fragment AdMLP de 64 mer contenant une séquence TATA (colonnes 5 et 6) ou d'un fragment d'ADN Pvul pSK endommagé par le cisplatine (respectivement 10 et 50 ng) (colonnes 7 et 8) non marqué par rapport à un fragment non endommagé (comparer avec les colonnes 9 et 10). Le fait que l'addition d'un fragment contenant une séquence TATA (TATA) a pour effet une compétition avec la liaison du TBP à l'échantillon d'ADN endommagé par le cisplatine (voir la colonne 2 comparée aux colonnes 5 et 6) , soutient la conclusion que l'échantillon déplacé correspond à un complexe TBP/ADN. Ces conclusions sont soutenues par des tests supplémentaires de fixation sur membrane qui évaluent l' affinité relative de TBP pour une lésion d'ADN en présence de la séquence TATA normale. Le fragment F879 irradié aux UV (Figure 1B) est incubé avec du TBP en présence de l'élément TATA du gène AdMLP (TATA) . Les résultats sont illustrés à la Figure 4B qui confirme une association préférentielle de TBP avec l'ADN endommagé (concentration de TBP de 0.7 à 10 ng) et qui indique que des quantités nettement plus importantes du fragment de AdMLP sont nécessaires pour concurrencer la liaison du TBP avec 1 ng d'ADN endommagé aux UV et atteindre un niveau de liaison TATA/TBP similaire à celui observé en l'absence d'ADN endommagé (la colonne indique de 10 à 200 ng de concentration pour le compétiteur de l'élément TATA). Comme contrôle, on utilise le TBP inactivé préalablement incubé pendant 15 min à 47°C (Nakajima et al. 1988) et qui ne montre aucune interaction, que ce soit avec l'ADN non-endommagé ou l'ADN endommagé aux UV.EXAMPLE 4: Migration on acrylamide gel (Fig 4). The ability of purified TBP to bind damaged DNA has also been observed in tests for variation of electrophoretic mobility using the EMSA technique. For that, we incubates a DNA probe (36 mer, 0.5 ng, 10,000 cpm) labeled with phosphorus 32 not damaged or containing only one cisplatin site (1,3-GpTpG) with recombinant TBP and in this second case, the formation of a complex with reduced electrophoretic mobility (20 ng) compared to the free probe is detected on acrylamide gels. In agreement with the membrane fixation tests, the EMSA technique has demonstrated the formation of a TBP / DNA nucleoprotein complex specific for damaged DNA. In the presence of TBP, a significantly larger amount of the damaged DNA sample is mobilized compared to the amount mobilized when the sample is not damaged (Fig 4A, compare columns 2 and 12). In addition, it can be seen that the formation of a damaged TBP / DNA complex is reduced significantly when increasing amounts (respectively 10 and 50 ng) of a 64-mer AdMLP fragment containing a TATA sequence are added (columns 5 and 6) or of a fragment of Pvul pSK DNA damaged by cisplatin (respectively 10 and 50 ng) (columns 7 and 8) unlabeled relative to an undamaged fragment (compare with columns 9 and 10). The fact that the addition of a fragment containing a TATA sequence (TATA) results in competition with the binding of TBP to the DNA sample damaged by cisplatin (see column 2 compared to columns 5 and 6) , supports the conclusion that the displaced sample corresponds to a TBP / DNA complex. These conclusions are supported by additional membrane binding tests that assess the relative affinity of TBP for DNA damage in the presence of the normal TATA sequence. The UV irradiated fragment F879 (FIG. 1B) is incubated with TBP in the presence of the TATA element of the AdMLP gene (TATA). The results are illustrated in FIG. 4B which confirms a preferential association of TBP with the damaged DNA (TBP concentration of 0.7 to 10 ng) and which indicates that significantly larger quantities of the fragment of AdMLP are necessary to compete with the binding of the TBP with 1 ng of DNA UV-damaged and reach a level of TATA / TBP binding similar to that observed in the absence of damaged DNA (the column indicates from 10 to 200 ng of concentration for the competitor of the TATA element). As a control, the inactivated TBP previously incubated for 15 min at 47 ° C. (Nakajima et al. 1988) is used and which shows no interaction, either with non-damaged DNA or DNA damaged with UV.
L' ensemble des résultats démontre clairement pour la première fois que deux types de lésions d'ADN, induites soit par irradiation aux UV, soit par traitement à la cisplatine, servent de cibles pour la liaison de TBP. La liaison de TBP est fonction du nombre de lésions présentes dans l'ADN et semble suffisamment forte pour persister en présence de quantités significatives de la séquence TATA. L' inhibition de la transcription et la rétention préférentielle de TBP sur une membrane de nitrocellulose ont été également observées avec un ADN endommagé à l' acétylaminofluorée (AFF) une aminé aromatique connue pour ses capacités carcinogènes .The overall results clearly demonstrate for the first time that two types of DNA damage, induced either by UV irradiation or by treatment with cisplatin, serve as targets for TBP binding. TBP binding is a function of the number of lesions present in DNA and appears to be strong enough to persist in the presence of significant amounts of the TATA sequence. Inhibition of transcription and preferential retention of TBP on a nitrocellulose membrane have also been observed with DNA damaged by acetylaminofluorine (AFF), an aromatic amine known for its carcinogenic capacities.
En outre, les résultats de la présente invention mettent en évidence l'affinité préférentielle de TBP pour les régions d'ADN endommagées présentant une courbure. Le procédé de criblage de produits cytotoxiques peut ainsi être employé pour discriminer plus particulièrement les produits qui induisent une courbure de l'ADN. Une telle classification est utile afin de déterminer le potentiel toxique et le mode d'action de nombreuses molécules cytotoxiques . Furthermore, the results of the present invention demonstrate the preferential affinity of TBP for damaged DNA regions exhibiting curvature. The method of screening for cytotoxic products can thus be used to discriminate more particularly the products which induce a curvature of the DNA. Such a classification is useful in order to determine the toxic potential and the mode of action of many cytotoxic molecules.
LISTE DE SEQUENCESLIST OF SEQUENCES
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D'ADN ENDOMMAGE DANS UN ECHANTILLON D'ADN ET SONDAMAGED DNA IN A SAMPLE OF DNA AND ITS SOUND
APPLICATION AU CRIBLAGE DE PRODUITS CYTOTOXIQUES AGISSANT SUR L'ADNAPPLICATION TO THE SCREENING OF CYTOTOXIC PRODUCTS ACTING ON DNA
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TCTTCTTCTT CTTCTGTGCA CTCTTCTTCT CT 32 BIBLIOGRAPHIETCTTCTTCTT CTTCTGTGCA CTCTTCTTCT CT 32 BIBLIOGRAPHY
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Claims

REVENDICATIONS
1. Procédé de mise en évidence et/ou dosage d'ADN endommagé dans un échantillon, caractérisé en ce qu' on évalue l' importance de la fixation du TBP ou d'un produit analogue sur l'ADN de l'échantillon et on détermine les résultats directement ou par rapport à un témoin.1. Method for detecting and / or assaying damaged DNA in a sample, characterized in that the importance of fixing TBP or a similar product on the DNA of the sample is evaluated and determines the results directly or in relation to a control.
2. Procédé selon la revendication 1, caractérisé en ce que l'importance de la fixation du TBP sur l'ADN est évaluée par détermination de la transcription d'un ADN non endommagé connu contenant un promoteur, dans un milieu assurant la transcription, la transcription n'étant assurée que par la présence dans ledit milieu de TBP résiduel non fixé sur l'ADN.2. Method according to claim 1, characterized in that the importance of the binding of TBP to the DNA is evaluated by determining the transcription of a known undamaged DNA containing a promoter, in a medium ensuring the transcription, the transcription being ensured only by the presence in said medium of residual TBP not attached to the DNA.
3. Procédé selon la revendication 2, caractérisé en ce que : a) l'échantillon est mis en incubation avec du TBP ou un produit analogue ; b) après incubation, on ajoute à l'échantillon une séquence d'ADN non endommagé connu contenant un promoteur, dans des conditions assurant la transcription mais sans TBP additionnel ; c) on détermine la transcription de cet ADN connu et ; d) on mesure la variation de transcription de cet ADN connu par rapport à une transcription normale ou par comparaison avec un témoin.3. Method according to claim 2, characterized in that: a) the sample is incubated with TBP or a similar product; b) after incubation, a known undamaged DNA sequence containing a promoter is added to the sample, under conditions ensuring transcription but without additional TBP; c) determining the transcription of this known DNA and; d) the variation in transcription of this known DNA is measured with respect to a normal transcription or by comparison with a control.
4. Procédé selon la revendication 3, caractérisé en ce que l'étape a) est effectuée dans des conditions qui ne permettent pas la réparation de l'ADN.4. Method according to claim 3, characterized in that step a) is carried out under conditions which do not allow the repair of DNA.
5. Procédé selon la revendication 3 ou 4 , caractérisé en ce que l'étape b) est effectuée dans un milieu constitué d'extraits cellulaires totaux.5. Method according to claim 3 or 4, characterized in that step b) is carried out in a medium consisting of total cell extracts.
6. Procédé selon la revendication 5, caractérisé en ce qu'il s'agit d'extraits cellulaires HeLa. 6. Method according to claim 5, characterized in that it is HeLa cell extracts.
7. Procédé selon la revendication 3 ou 4, caractérisé en ce que l'étape b) est effectuée dans un milieu de transcription reconstitué (RTS) .7. Method according to claim 3 or 4, characterized in that step b) is carried out in a reconstituted transcription medium (RTS).
8. Procédé selon la revendication 1, caractérisé en ce que ledit échantillon est mis en présence d' un réactif contenant du8. Method according to claim 1, characterized in that said sample is placed in the presence of a reagent containing
TBP ou un produit analogue et on évalue l' importance du dommage de l'ADN par mise en évidence et/ou dosage du complexe TBP/ADN formé directement ou par rapport à un témoin.TBP or a similar product and the extent of DNA damage is evaluated by detecting and / or assaying the TBP / DNA complex formed directly or in relation to a control.
9. Procédé selon la revendication 8, caractérisé en ce que la formation du complexe TBP/ADN est mise en évidence et/ou dosée par : a) fixation dudit complexe sur un support ; b) variation de la mobilité dudit complexe sur gel ; c) empreinte à la DNase I. 9. Method according to claim 8, characterized in that the formation of the TBP / DNA complex is demonstrated and / or measured by: a) fixing of said complex on a support; b) variation of the mobility of said complex on gel; c) DNase I fingerprint.
10. Procédé selon l'une des revendications 8 ou 9, caractérisé en ce que la formation dudit complexe est évaluée par la mesure de la quantité d'ADN retenue sur une membrane de nitrocellulose .10. Method according to one of claims 8 or 9, characterized in that the formation of said complex is evaluated by measuring the amount of DNA retained on a nitrocellulose membrane.
11. Procédé selon l'une des revendications 8 ou 9, caractérisé en ce que la formation dudit complexe est évaluée par la variation de mobilité du complexe sur un gel d' acrylamide .11. Method according to one of claims 8 or 9, characterized in that the formation of said complex is evaluated by the variation in mobility of the complex on an acrylamide gel.
12. Procédé selon la revendication 11, caractérisé en ce que l' évaluation de la variation de mobilité est effectuée par la technique EMSA.12. Method according to claim 11, characterized in that the evaluation of the variation in mobility is carried out by the EMSA technique.
13. Procédé selon l'une des revendications 1 à 12, caractérisé en ce qu'on utilise du TBP purifié sous forme libre.13. Method according to one of claims 1 to 12, characterized in that TBP purified in free form is used.
14. Procédé selon l'une des revendications 1 à 12, caractérisé en ce qu'on utilise du TBP lié au facteur FHIID. 14. Method according to one of claims 1 to 12, characterized in that TBP linked to the FHIID factor is used.
15. Application du procédé selon l'une des revendications 1 à 14 à la mise en évidence et/ou au criblage de produits cytotoxiques sur l'ADN.15. Application of the method according to one of claims 1 to 14 to the detection and / or screening of cytotoxic products on DNA.
16. Application selon la revendication 15 au criblage de produits anticancéreux. 16. Application according to claim 15 for the screening of anticancer products.
17. Application du procédé suivant l'une des revendications 1 à 14 pour le suivi thérapeutique d'un traitement anticancéreux .17. Application of the method according to one of claims 1 to 14 for the therapeutic monitoring of an anticancer treatment.
18. Application du procédé selon l'une des revendications 1 à 14 à la mise en évidence et/ou au criblage de produits cytotoxiques sur l'ADN, caractérisée en ce qu'on fait réagir au préalable ledit produit dans l'échantillon d'ADN non endommagé, ledit échantillon étant alors dépourvu de tout autre produit susceptible d'endommager l'ADN et la cytotoxicité du produit est établie lorsqu'on détecte de l'ADN endommagé. 18. Application of the method according to one of claims 1 to 14 to the detection and / or screening of cytotoxic products on DNA, characterized in that said product is reacted beforehand in the sample of Undamaged DNA, said sample then being devoid of any other product capable of damaging the DNA and the cytotoxicity of the product is established when damaged DNA is detected.
PCT/FR1998/000339 1997-02-21 1998-02-20 Method for the isolation and/or assay of damaged dna and its application to screening of cytotoxic products acting on dna WO1998037233A1 (en)

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US6913880B1 (en) 1998-10-09 2005-07-05 Xgene Corporation Method for determining transcription factor activity and its technical uses
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