WO1990004650A1 - Procede de purification pour complexes d'adn/proteines lies par covalence - Google Patents

Procede de purification pour complexes d'adn/proteines lies par covalence Download PDF

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WO1990004650A1
WO1990004650A1 PCT/GB1989/001263 GB8901263W WO9004650A1 WO 1990004650 A1 WO1990004650 A1 WO 1990004650A1 GB 8901263 W GB8901263 W GB 8901263W WO 9004650 A1 WO9004650 A1 WO 9004650A1
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dna
protein
peg
topoisomerase
phase
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PCT/GB1989/001263
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Derek Fisher
Gillian Elizabeth Francis
Robert Anderson
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Royal Free Hospital School Of Medicine
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • 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
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

Definitions

  • the present invention relates to processes for purifying and estimating covalent DNA/protein complexes and to materials produced by the process.
  • Phase systems of immiscible aqueous solutions of polymers and/or salts provide a means of separating cells an macromolecules on the basis of their differential affinities for the two component phases [Walter H, Brooks DE, & Fisher D. (eds.) Partitioning in aqueous two-phase systems, theory, methods, uses and applications in biotechnology. Academic Press, Orlando 1985].
  • covalently bound DNA/protein complexes may be separated from non-covalently bound DNA/protein complexes and unbound DNA using a suitable aqueous biphasic system comprising a top phase of PEG solution and a bottom, phosphate solution phase.
  • PEG polyethylene glycol
  • the present invention therefore provides a process for separating covalent DNA/protein complexes from non- covalent DNA/protein complexes and unbound DNA comprising th steps of:
  • step (ii) subjecting the product of step (i) to phase partitio between an aqueous PEG solution phase and an aqueous phosphate solution phase.
  • covalently bound DNA/protein complexes ar recovered from the aqueous PEG phase.
  • Coupling of PEG to the DNA/protein complexes is preferably conducted by reacting the complexes with a reactive 2,2,2-trifluoroethanesulphonyl (tresyl) derivative of PEG, preferably tresyl monomethoxyPEG (TMPEG) which is described in our British Application No. 8824591.5.
  • tresyl 2,2,2-trifluoroethanesulphonyl
  • TMPEG tresyl monomethoxyPEG
  • Fig. 1. shows schematically the coupling of PEG to DNA/protein complexes.
  • Fig. 2. is a graph showing the effect of sonication on the molecular weight of salmon sperm DNA, (12 ⁇ peak to peak amplitude, 20kHz15OW) .
  • Fig. 3. is a graph showing the effect of sonication on protein-free DNA partitioning.
  • Fig. 4. shows graphically the effect of extra rounds of phosphate extraction on recovery of DNA from the PEG phase.
  • Fig. 5 and Fig. 6 show the dose related increment in PEG phase DNA following treatment of cells with VP-16-213 (Fig 5 and UV (Fig.6) .
  • Fig. 7, 8, 9, 16 and 21 shows scanning densitometer plots.
  • Fig. 10. is a graph of yields from PEG phase separation of Trask preparations.
  • Fig. 11. shows differential DNA recovery from retinoic acid induced and uninduced cells.
  • Figs. 12 and 13 shows results of differential hybridisation experiments.
  • Figs. 14 and 15 shows results of differential hybridisation experiments.
  • Figs. 17 and 18 shows the effect of retinoic acid and novobiocin on yield of DNA produced by the Trask method (Fig. 17) and the present method (Fig. 18) .
  • Figs. 19 and 20 shows tubulin hybridisation to fractionated DNA from various induced or uninduced cells.
  • Figs. 22 and 23 show hybridisation signals from cloned DNA prepared by the process of the invention.
  • Fig. 24 shows increments in PEG phase DNA from induced or uninduced cells exposed to VP16.
  • Fig. 25 shows the effect of m-AMSA on DNA yield when partitioned according to the invention.
  • Fig. 26 shows the effect of VP16 on PEG-phase yield from cells pretreated with retinoic acid.
  • Fig. 27 shows the use of UV cross-linking to demonstrate retinoic acid induced DNA protein association.
  • the process of the invention exploits two phenomena (shown schematically in Figure 1) .
  • activation of PEG with tresyl chloride (panel 1, Figure 1) allows the PEG to b covalently attached to proteins (panel 2) .
  • Panel 2 This gives DNA/protein complexes very high affinity to the PEG-rich top phase, allowing the attached DNA to be recovered, while unattached DNA remains in the phosphate-rich bottom phase (panel 5) . This can be used to recover or quantitate the protein-linked DNA.
  • the level of DNA in the top (PEG) phase is dependent on the level of DNA/protein complexes in the sample, on the average size of DNA attached to protein (the latter is controlled for some applications by sonication, DNAse treatment, or restriction endonuclease digestion of th sample) .
  • reaction mixture and/or phase partitioning conditions described here result in the non- covalently bound protein (with which the majority of eukaryotic DNA is associated) becoming detached from the DNA
  • the method therefore results in DNA that was unbound or non- covalently bound to protein being left in the phosphate phase.
  • PEG is known to compact and alter the conformation o DNA.
  • the present invention may be exploited in, for instance, the following:
  • the invention further provides products comprising a DNA fragment covalently bound to a protein having at least one PEG moiety bound to the protein.
  • the PEG moiety is a monomethoxy PEG group.
  • This tresyl-PEG preparation (TMPEG) was washed to remove pyridine before use.
  • the TMPEG was dissolved in methanol-HCl mixture (250:1) and allowed to precipitate -20° for 8 h.
  • the resulting white solid was collected at 0°C and the filtrate checked for pyridine content spectrophotometrically ( . max 255 nm) .
  • This procedure was repeated by using methanol-HCl (1000:1) as washing mixture until no pyridine could be detected.
  • the pyridine- free TMPEG (10-12g; 55-66% yield) was dried under vacuum for several hours at room temperature. Sulphur content was 0.5% (theoretical content for 1 tresyl group per molecule of MPEG 5000 is 0.62%).
  • the TMPEG was stored desiccated at 4°C prio to use.
  • TMPEG 400mg/ml in 0.05M Na phosphate 0.125 NaCl buffer pH7.5 was mixed with cell lysate or DNA/protein complexes at a ratio 1:1 (v:v) on a rotating mixer for 2h at room temperature. Since we do not know the number of lysine molecules per molecule of topoisomerase II and there are other proteins present, we chose to add TMPEG in excess. Experiments with albumin indicate maximum partitioning to th PEG phase with a TMPEG:lysine molar ratio 8:1 and significantly increased partitioning was achieved at much lower values.
  • the ratios of DNA/protein complexes to TMPEG used in these experiments therefore represent a gross excess of TMPEG, but this may have additional advantageous effects because of the effects of PEG concentration on DNA conformation (4,5). Further experiments will be needed to evaluate these two facets of the separation procedure independently (using mixtures of TMPEG and PEG during the covalent modification step) .
  • TMPEG treated material Aliquots of TMPEG treated material were added directly to the phase system. Where further enzymatic treatments (e.g. with restriction enzymes) are planned it ma be advisable to neutralise unreacted TMPEG to prevent it modifying these proteins.
  • enzymatic treatments e.g. with restriction enzymes
  • lysine or albumin can be used to prevent further undesired reaction of the TMPEG after the coupling step.
  • proteinase K is robust and, provided overnight incubations 37°C are used, is little affected by the addition of TMPEG- treated material.
  • the phase system chosen consists of 10% (w/w) poly(ethylene glycol) 6000 (BDH). 14% phosphate (ratio of 16.86g KH 2 P0 4 and 40.20g K 2 HPO 4 (3H 2 0) and 76% distilled deionised sterile water.
  • BDH poly(ethylene glycol) 6000
  • phosphate ratio of 16.86g KH 2 P0 4 and 40.20g K 2 HPO 4 (3H 2 0) and 76% distilled deionised sterile water.
  • the system was allowed to mix and settle into an upper PEG-rich and a lower phosphate-rich phase. These were separated and individually filtered through 0.22um filter (Gelman Science Inc. Michigan) aliquotted and stored at -20°C. This was done because it is difficult to sample aliquots of the mixed phase system without taking varied proportions of the two phases and this would reduce the reproducibility of the method.
  • the phase system was reconstructed usually using the stated volume ratio of PEG:phosphate phases, with no more than 15% of the total volume of the phase system consisting of tresyl-PEG treated DNA/protein extract.
  • the ratio of P0 4 :PEG phases was increased usually to 750:250ul and multiple rounds of phosphate extraction were used.
  • the phases were mixed by vortexing and allowed to settle at 25°C for 10 min.
  • the PEG rich phase was then transferred to a fresh phosphate phase and the procedure repeated (the number of rounds of extraction is indicated with each experiment) .
  • DNA/protein complexes equivalent to 2.5ug DNA were exposed to lOug DNase I (bovine pancreatic DNAse, Sigma Ltd.) in 500ul of reaction mixture.
  • the latter contained 50M Tris HC1; 25mM MgCl 2 ; 20mM Cl; ImM CaCl 2 ; 10% w/v glycerol; 50ug/ml bovine serum albumin (BSA - Sigma Ltd.). After the exposure times indicated, the reaction was stopped using a final concentration of 1% SDS and 15mM EDTA.
  • the level of DNA in the PEG phase serves as a measur of the DNA complexed to topoisomerase, but this cannot readily be measured fluorimetrically in the presence of the protein because of quenching. Phenol/chloroform extraction is used to separate protein from DNA, but cannot be used directly because protein covalently linked to DNA will cause loss of the attached DNA (usually the DNA desired) .
  • the topoisomerase must therefore be digested. Proteinase K (Sigma Chemicals Poole) 20mg/ml aqueous solution was added (lOOul/ml of PEG or phosphate phase) and incubated at 37°C overnight. This step can then be followed by an extraction procedure to recover the DNA (e.g.
  • the Sephadex was pre-equilibrated with lOmM Tris HC1, lOOmM NaCl ImM EDTA pH 8.0 (STE) . Columns were spun at 400g for 4 min. DNA was recovered from the eluate by precipitation in 2 volumes of absolute alcohol at -70°C for 1 hour followed by centrifugation at ll,000g for 1 min. in a microfuge (MSE instruments) and the pelleted DNA resuspended in 20ul of Tris EDTA: lO M Tris:HCl, ImM EDTA pH7.4 (TE) . This DNA can then be estimated by conventional means.
  • the D A was estimated from the integrals of scanning densitometer plots of the negatives (Joyce Lobel Instruments Gateshead) b comparison with know amounts of D ⁇ A. Experiments adding known amounts of D ⁇ A individually to PEG and phosphate phase with this procedure showed that recoveries vary for the two phases. For a rapid uantitation samples were spotted onto an agarose slab, photographed on a transilluminator as above and compared to a set of D ⁇ A standards.
  • HL60 cells in log phase growth were harvested by centrifugation at 400g for 6 min. and resuspended in serum free medium (RPMI Gibco Ltd.). at 5 x 10 6 eelIs/ml.
  • Cells were lysed in the presence of protease inhibitors by the addition of 10% aqueous SDS to a final concentration of 1% with 1% v/v Triton X-100 (Sigma) , 15mM EDTA and ImM phenylmethylsulphonylfluoride (Sigma) from a stock solution of lOOmM in methanol. The cell lysate was vortexed aliquotted and stored at -20°C.
  • Etoposide (VP 16-213 Bristol Meyers, NY state USA) was serially diluted in serum free RPMI with an appropriate diluent control, to final concentrations of 10 ⁇ 5 M down to 10 ⁇ 9 M and an equal concentration of diluent throught the gradient. This was then allowed to react with the cells for 15 ins at 37°C. The reaction was finally stopped by the addition of sodium dodecyl sulphate and Triton X-100 (both Sigma Chemicals - Poole England) to a final concentration of 1% (v/v) . Phenyl methyl sulphonyl fluoride (PMSF) and disodium ethylenediaminotetraacetic acid (EDTA both Sigma Chemicals - Poole England) were also added at a final concentration of ImM and 15mM respectively.
  • PMSF Phenyl methyl sulphonyl fluoride
  • EDTA disodium ethylenediaminotetraacetic acid
  • Tresyl monomethoxy poly(ethylene)glycol (TMPEG) was dissolve in coupling buffer (0.5M NaP04 0.08M NaCl pH 7.5) to a concentration of 400mg/ml and an equal ratio (200ul: 200ul) was allowed to react with each of the cell lysates on a rotating turntable at room temperature for two hours.
  • the cells were resuspended in 200 microlitres 10 mM Tris HCl pH 7.4 ImM EDT (T.E.) sodium dodecyl sulphate, SDS (Sigma) and Triton X - 100 (BDH) to a final concentration of 1% and disodium EDTA (Sigma) was added to a final concentration of 15 mM.
  • the samples were then cooled to 4°C and sonicated with an MSE bench sonicator on ice for 30 seconds, 12 micrometers amplitude peak to peak, 150 Watts and 20 kHz. This reduces the modal size of the DNA to about 800 basepairs.
  • the negative control sample was incubated at 56°C for 1.5 hours with proteinase K (Sigma) to a final concentration of 5 micrograms per microlitre. This sample was then treated as the others.
  • Tresyl methoxypoly(ethylene glycol) TMPEG was prepared to 400 mailgrams per millilitre buffer in 0.05 sodium phosphate 0.125 sodium chloride pH 7.5 and was mixed
  • phase system composed of 750 microlitres phosphate-rich and 250 microlitres PEG-rich phases.
  • the system was constructed using 14.7 % w/w poly (ethylene glycol) 6000 (BDH), 11.2% w/w phosphate (ratio of 16.86 gram KH2P04 and 40.20g K2HP04 (3H2o) both BDH) and 74.1% distille deionised sterile water.
  • the phases were vortexed and allowed to settle for 10 minutes at 25°C after which the upper PEG-rich phases were removed to fresh 750 microlitre phosphate rich bottom phases. This was then repeated to leave one top phase and four bottom phases per condition.
  • the phosphate is removed spinning a sample down a 1 ml syringe packed with Sephadex G-50 (Pharmacia) which has been equilibrated with STE pH8 (lOmM Tris HCl pH8; ImM EDTA;100mM NaCl) .
  • STE pH8 lOmM Tris HCl pH8; ImM EDTA;100mM NaCl
  • the eluate is then precipitated with two volumes of cold absolute alcohol at -70*C overnight.
  • the DNA was then spun for 10 minutes at 11,000 r.p.m in a MSE microcentrifuge and resuspended in 10 microlitres T.E. pH 7.4. This was then run on a 1% agarose gel for 2 hours at 2.5 volts per centimetre. The gel was photographe on a UV trans-illuminator with Polaroid 665 film. The negatives scanned with a densitometer (Joyce Lobel) and the integrals were used to calculate the amount of DNA present using a known salmon sperm standard.
  • HL60 cells were pre-incubated with 10 ⁇ 3 M novobiocin (Sigma Ltd.) for 60 min at 37°C under standard culture conditions (see above) and were then either exposed to retinoic acid (10" 6 M for 70 min) or sham treated with diluent. Toxicity tests showed that less than 5% cells died with this dose of novobiocin when assessed by nigrosin.
  • Novobiocin is an established inhibitor of DNA topoisomerase II that acts in a different manner to the epipodophyllotoxins and intercalators, in that, unlike the latter it does not stabilise cleavable complexes of enzyme and DNA, but tends to inhibit binding of the enzyme to DNA.
  • the agent has a complex interaction with the enzyme in that with alterations in the ratio of drug:enzyme both inhibitory and stimulatory effects can be observed (Collins and Johnson Nucl Acids Res 7:1331;1979)
  • the filters were prepared by the dot blot method of FC Kafatos, CW Jones and A. Efstratiadis. Nuclec Acid Research 7:1541;1979. Lifted from The Guide to Molecular Cloning Techniques, edited by S.L. Berger and A.R. Kim el Academic Press 1987. 1) Preparation of nitocellulose or nylon GeneScreen Plus filters (New England Nuclear Research Products - Boston USA) with DNA from DNA/protein complexes recovered from cells induced by all trans retinoic acid or phorbol-12-myristate 13-acetate (PMA) (both Sigma Chemicals - Poole England) .
  • PMA phorbol-12-myristate 13-acetate
  • Ethylenediaminetetraacetic acid, EDTA Ethylenediaminetetraacetic acid, EDTA
  • Eac sample then had addition of TE pH 8 to bring the volume to a standard 50ul. All the samples were heated to 70°C with 0.1 volume of 3M sodium hydroxide (BDH Chemicals - Poole England for 45 minutes. This denatured the DNA and destroyed any RNA, if present. The samples were then allowed to cool to room temperature and 55ul 2M ammonium acetate (BDH Chemicals - Poole England) was added. Serial dilutions were then carried in 1M ammonium acetate.
  • 3M sodium hydroxide BDH Chemicals - Poole England
  • the DNA was loaded onto the nylon (or nitro cellulose) with a "home made 1 Dot - blot device and allowed to stand at room temperature for at least 30 minutes prior to being sucked through by vacuum. Filters were then dried between 3MM paper (Whatman Paper - Maidstone England) and then baked for two hours at 80°C under a vacuum (optional for nylon filters) .
  • the filters were then pre- hybridised, using 6x SSC, (0.9M Saline, 0.9M Sodium Citrate pH 7.0 Both BDH Chemicals - Poole England), 0.5% SDS, (Sodiu Dodecyl Sulphate, Sigma Chemicals - Poole England) 5x Denharts olution (0.5g Ficol, 0.5g polyvinylpyrrolidone and 0.5g Bovine Serum Albumen Pentax Fraction V - all Sigma Chemicals, Poole England) and lOOmcg/ml of highly sonicated salmon sperm DNA (Sigma Chemicals - Poole England) in 200mls of distilled deionized water.
  • 6x SSC 0.9M Saline, 0.9M Sodium Citrate pH 7.0 Both BDH Chemicals - Poole England
  • SDS Sodiu Dodecyl Sulphate
  • 5x Denharts olution 0.5g Ficol, 0.5g polyvinylpyrrolidone and 0.5g Bovine Serum
  • the filters were prehydridise for 3.5 hours at 68°C, with agitation, in the shaking waterbath. After pre-hybridisation, the fluid was poured of and hybridisation fluid was added which consisted of 6x SSC,0.5% SDS, Denharts, 100 mcg/ml denatured salmon sperm DN and 0.1M EDTA in 100ml volume. To this was added a probe radio labelled with dCT 3 p, using the random primer method kit (Amersham Pic - Amersham England) and the amount of incorporation was assessed using adsorption to Whatman DE-81 filter paper Manniatis loc.cit. with washing to remove free nucleotides.
  • Hybridisation was allowed to proceed again at 68°C with agitation for about 17 hours.
  • Post hybridisational washes were twice 45 minutes and then twice 30 minutes of lx SSC, lx Denharts and 0.1% SDS in 100ml distilled deionized water which had been pre warmed to 68°C. This solution was added to the filters after the hybridisation fluid had been removed.
  • the post hybridisational washes were also carried out with agitation in the waterbath. These washes were then followed by two final 20 minute rinse washes also with agitation in the waterbath with 100ml of pre warmed O.lx SSC at 68°C.
  • the filters were then blotted between sheets of Whatman 3MM paper and then allowed to dry thoroughly in air.
  • the filters had a count of no more than 2-5 cpm above background and were exposed to Fuji RX 100 medical X - ray film (Fuji Photo Film Co. Ltd. - Japan) with two rare earth intensifying screens (Du Pont Cronex Quanta III, Du Pont Industries U.K.) at -70°C for 24-48 hours depending upon the result obtained.
  • This material (5.0ug) was then exposed to 0.05 ug Bovine Pancreatic DNase 1 (Sigma Chemicals - Poole England) for a period of five minutes in the presence of 50mM Tris.Cl pH 8.0, 10 % v/v Glycerol, 0.1% Bovine Serum Albumen, 250mM Mg 2+ lOmM Ca 2+ and 200mM K ⁇ all Sigma Chemicals Poole - England) in 250ul volume.
  • a similar probe (R+) was prepared form analogous, undifferentited HL60 cells.
  • the reaction was stopped by bringing the reaction mixture up to 1% SDS and 15mM EDTA.
  • An equal volume of 400mg/ml TMPEG (tresyl monomethoxy polyethylene glycol) was allowed to react to the protein present for two hours at room temperature and then was partitioned on a 250/270 ul PEG:P0 4 phase system with five rounds of P0 4 .
  • the DNA was then liberated from the protein by the addition of proteinase K (Sigma Chemicals - Poole England) to 20mg/ml overnight at 37°C.
  • the samples were then extracted with two rounds of phenol/chloroform followed by a single extraction with isoamyl alcohol/chloroform.
  • topoisomerase-associated DNA was as follows: the overhang created by topoisomerase II cleavage is endfilled with Kleno (Amersham U.K.) and then Sa I linkers (Pharmacia) ligated to the blunt ends. The DNA is then digested with Eco RI and Sa I (Eco RI to digest the largest DNA fragments down to a reasonable size and Sal I to create sticky ends) . The DNA i then ligated to pUC 18 which has been digested with Eco RI and Sal I and transformed into E.coli DH5 cells (BRL) . This procedure should clone both randomly cleaved ends of DNA as well as the topoisomerase associated DNA, but will tend to exclude Sal I/Sal I fragments cut from sections of DNA distant from the topoisomerase II cleaage site.
  • the DNA fractionated by the new method initially proved resistant to digestion with restriction enzymes however this was over come by first passing the DNA through QIAGEN (Trademark) tips.
  • the DNA for cloning by this procedure should be kept as large as possible to reduce the number of randomly cleaved ends available for cloning.
  • Cloning may be impoved by using vector cut with Sal I alone and then treated with calf intestinal phosphatase to remove terminal phosphate groups. If there is a Sal I site between the artificially created Sal I site at the topoisomerase cleavage site by the attachment of a Sal I linker and the nearest Eco RI site, then these will be cloned (as will as other Sal I-Sal I fragments) .
  • IPTG isopropyl-thiogalactopyranoside
  • Figs 2&3 shows the influence of sonication on PEG- phase yield. As the molecular weight of DNA falls the yield in the PEG phase increases.
  • Figure 3 shows the influence of sonication on Peg phase yield using 1:1 PEG:phosphate phase volume ratio and a single round of partitioning (circles) or seven rounds with fresh phosphate phases (squares) .
  • Figure 4 shows the effect of additional rounds of extraction with phosphate phase on the recovery of DNA in the PEG and phosphate phases. Results are means+SEM of 4 independent experiments.
  • Proteinase K treated aliquots of similarly fractionated samples were processed identically to determine what proportion of the recovered PEG-phase DNA was protein linked (Table I)
  • VP-16 (15 min exposure) of whole cells was used to increase the level of topoisomerase II complexed to DNA.
  • the results are eans+SEM of PEG-phase yields for 8 independent experiments, expressed with respect to untreated controls. (Fig. 5).
  • the amount of DNA complexed to topoisomerase II in the preparation to be fractionated was varied by exposing HL60 cells to increasing concentrations of VP16-213. This drug stablises the enzyme at the stage where it is covalently bound to DNA (11) and prevents completion of the reaction (the ligation and dissociation steps) .
  • Figure la shows a dose-related increase in DNA recovery from the PEG phase maximal at 10 ⁇ 5 M VP16-213. Phase partitioning was performed as described below (multiple phosphate extractions were not used in these experiments) . Results are means +SEM of 8 independent experiments and are expressed as a % of the amount of DNA recovered without exposure of cells to VP16- 213. In 2/8 experiments the sample was sonicated prior to fractionation. An increment at 10 ⁇ 9 M VP16-213 was detected in both the sonicated and 3 of the 6 unsonicated samples, otherwise responses were similar.
  • UV cross linking was used to increase the level of protein covalently bound to DNA.
  • the results are means+SEM of 3 independent experiments expressed with respect to untreated controls. (Fig. 6) .
  • UV radiation is well known to induce covalent crosslinking between proteins and DNA. This example is relevant to the adaptation of the method for the examination of proteins that bind to DNA non-covalently with the method (also further discussed in and example below) .
  • the new method may, since it can partition DNA to which a single topoisomerase molecule is attached, be of advantage for the preparation of differentiation site DNA. This does not of course mean that the Trask procedure cannot be used in series with the new technique to produce a two- step fractionation procedure for differentiation-associated topoisomerase cleavage sites, since even if the efficiency o precipitation is low, differentiation sites with one or few topoisomerase molecules are not likely to be less represente in the precipitate than in the supernatant.
  • fragments in this size range should include some that were attached to a single enzyme molecule (see below) .
  • the differential recovery suggests several possibilities: an increase in the ratio of protein-bound DNA to free in SDS/KC precipitates from differentiating versus undifferentiated cells (anticipated on the basis of the known actions of DNA topoisomerase II in differentiation) however we cannot exclude: a qualitative change in the types of proteins bound vis a vis their accessible lysyl residues for PEG- modification; or a decreasse in the average DNA fragment siz of the SDS/KCL precipitates (since with longer fragments it is more difficult to overcome the affinity to the phosphate phase) .
  • This latter hypothesis seems unlikely, since, although high resolution electrophoresis of SDS/KCL precipitates has not yet been performed, no dramatic reduction of these fragments below 20Kbp seems to be occurring in differentiated cells.
  • Sonication and other methods to fragment DNA have two influences on the method. Opposing forces act on the DNA- protein complexes. If the DNA attached to a single molecule is long enough it could, via its affinity for the phosphate phase, retain the complex in that phase. Shortening the DNA will reduce the likelihood of this occurring. The second motive for shortening the DNA is if one wishes to recover the DNA at the protein binding site with little contamination by adjacent DNA.
  • the protein free DNA of the PEG phase is extractable by additional rounds of partitioning with fresh phosphate phases or by counter current distribution (i.e. chro atographic separation in a multiple phase partitioning apparatus) .
  • the experiment described in Example 1 and Fig. 4 demonstrates the efficiency of additional rounds of extraction with phosphate phase in removing PEG-phase DNA after sonication.
  • Sonication reduces the average length of the DNA in the DNA/protein preparation and concomitantly reduces the DNA partitioning to the PEG phase.
  • PEG-phase DNA was reduced to 32+17% of values in parallel experiments of unsonicated protein- linked DNA (using 4 different types of samples i.e. drug treated and controls) .
  • This modification of the method may be important in achieving detection of complexes where there is only a single isolated protein molecule bound to a section of DNA. It is also potentially useful in applying the method to cloning of DNA at protein binding sites, because sonication removes adjacent DNA.
  • FIG. 7 shows an example of affinity partitioning of small PEG/protein/DNA complexes. DNA/protein complexes were sonicated for 60 seconds to fragment the DNA. Topoisomerase II is know to protect DNA fragments of circa 140 base pairs in DNAse protection assays. Thus fragments of this size are unlikely to have more than single molecule of enzyme attached to the DNA.
  • nucleases e.g. DNAse and restriction endonuclease
  • DNAse digestion of DNA protein complexes prepared by the Trask method, using the protocol described in methods, after 10-20 minutes produces fragments comparable to 60 seconds sonication ( Figure 8) .
  • This method can also therefore be used to reduce DNA size for partitioning either before the treslation step ( Figure 9) , or after the first round of partitioning ( Figure 10) before subsequent partitioning or DNA cloning.
  • the protein K digested control in Fig 9 demonstrates that the recovered low molecular weigh DNA is the result of affinity partitioning since none is recovered if the PEG-protein is first removed from the DNA b proteinase K digestion.
  • the method can be used to prepare samples enriched for such fragments. This has applications in the analysis of DNA protein binding sites using the DNAse protection principle.
  • retinoic acid induces DNA topoisomerase II to perform DNA breakage-reunion reactions shortly after the induction of differentiation. This can be demonstrated in several ways (e.g. nucleoid sedimentation, the alkaline filter elution technique and the fluorescent alkaline DNA unwinding - FADU- technique) and is inhibited by DNA topoisomerase II inhibitors (13) . None of these methods is however suitable for the purification of these complexes.
  • the Trask procedure as mentioned above is likely on theoretical grounds to be inefficient for isolated protein molecules attached to DNA. It also does not in practice recover greater amounts of DNA/protein complexes from retinoic acid treated cells than from controls (see Examples 3 above) .
  • the Trask technique is unlikely therefore to be providing a significant enrichment of this type of DNA/protein complex vis a vis other complexes (the technique is known to be highly efficient for DNA/topoisomerase II complexes from DNA replication forks) .
  • Topo II-associated DNA from RA induced differentiating cells, labelled and hybridised this DNA to DNA enriched for, or depleted of, Topo II-bound DNA from both undifferentiated HL60 cells and cells induced to differentiate by RA or phorbol ester.
  • Differential hybridisation demonstrated that the isolated DNA is enriched for specific sequences.
  • Differential hybridisation was also seen with myc and fos probes suggesting that Topo II cleavage occurs near these genes. This is, as far as we are aware, the first demonstration that Topo II interacts at specific (or limited) sites in the genome during the induction of differentiation. It is important to note that although enhanced recovery is seen when topoisomerase II is arrested at the covalent complex stage by VP-16 (etoposide) , this agent is not used in the following experiments since it is known to influence cleavage site specificity.
  • DNAse digestion of DNA/Topo II complexes prepared by the Trask method for 1, 2 and 5 minutes demonstrates a clear protected band of DNA and this material can be affinity partitioned (see Examples above) .
  • Fig. 14 shows that similar differential hybridisation is achieved with independently prepared filter and probe samples (2 probe and 6 filter samples) .
  • PMA hybridisation also showed reproducible differential hybridisation.
  • 10/10 hybridisations there is a more intense signal from the R+ than R- DNA as shown in Figue 14.
  • UBSTITUTE SHEET events take place in or near the myc and fos genes during induced granulocytic differentiation. Since differential hybridisation was lost for myc when novobiosin was added (see Figure 16 other DNA protein linking events seem unlikely. Scanning densitometer traces of Fig. 16 show differential hybridisation to the myc probe by R+ and R- DNA is lost when R+ cells are pretreated with novobiocin (an inhibitor of Topo II) ; lines, A, B and C respectively.
  • Novobiocin produced no significant decrease in the amount of DNA recovered by the combined Trask/new procedure, neither in cells induced with retinoic acid nor in undifferentiated cells as shown in Figure 18. e) Demonstrations that DNA flanking stably expressed (differentiation unmodulated) genes is not enriched by the method.
  • the homopoly er tailing technique was used to introduce the prepared DNA fragments into dCTP tailed Plasmid (pUC8) . This method was selected because no differential handling of topoisomerase cleavage sites, sheared ends or restriction sites is involved (we wanted to avoid differences in efficiency of processing fragments with and without the topoisomerase staggered cleavage sites) . Transformation followed by growth of plasmid in, and recovery from DH5 E. coli, demonstrated that recombinant plasmids were produced. However the eficiency of this method was relatively low.
  • the second strategy was to infill the overhang created by the topoisomerase cleavage reaction using the Klenow polymerase and to attach Sal I linkers. After digestion with EcoRI and SA1 I, the DNA was ligated into pUcl ⁇ , similarly digested, and transformed into E. coli DH5 cells.
  • Clones were screened for differential hybridisation to R+ R- and whole genomic probes (i.e. using a similar strategy to that used above) , to determine DNA inserts which represent DNA sequences preferentially associating with topoisomerase during the induction of differentiation.
  • a sample of 23 of the first 298 clones obtained shown in Figures 22 and 23.
  • the combined hybridisation signal (absorbance per mm2) for three hybridisations to measured amounts of DNA from clones is sh shown in Fig. 22 R+ and R- probes were as for the differential hybridisation experiments above, WG was labelled whole genomic (unfractionated) HL60 cell DNA.
  • the hybridisation signals (absorbance per mm2) for three hybridisations, given in fig. 22 are expressed as a proportion of the combined hybridisation signal in Fig. 23.
  • the method can be used to quantitate the co plexing of topoisomerase to DNA induced or inhibited by different classes of topoisomerase II inhibitors.
  • the method is very sensitive for the detection of VP- 16, detecting a 326+100.25 %increase in PEG phase DNA at 10 ⁇ 9 M VP16 (means +SEM of 10 experiments using either undifferentiated or differentiation induced HL60. An increment (maximum 950%) was detected in 7/10 experiments. Increments in PEG phase DNA in the majority of samples at lO" 9 VP16-213 as shown in Fig. 24.
  • the method thus provides a method for discriminating topoisomerases with inherent or imposed different susceptibilities to inhibitor.
  • Weniger P An improved method to detect small amounts of radiation damage in DNA of eukaryotic cells. Int J Radiat Biol 36:197-199,1979.

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Abstract

On a mis au point un procédé de séparation de complexes d'ADN/protéines covalents de complexes d'ADN/protéines non covalents et d'ADN non lié comprenant les étapes consistant (i) à traiter les complexes d'ADN/protéines à l'aide d'un dérivé réactif de polyéthylèneglycol (PEG) et (ii) à soumettre la production de l'étape (i) à une séparation de phases entre une phase de solution de PEG aqueuse et une phase de solution de phosphate aqueuse.
PCT/GB1989/001263 1988-10-20 1989-10-20 Procede de purification pour complexes d'adn/proteines lies par covalence WO1990004650A1 (fr)

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WO1995006058A1 (fr) * 1993-08-24 1995-03-02 Polymasc Pharmaceuticals Plc Modification de polymere
US5446090A (en) * 1993-11-12 1995-08-29 Shearwater Polymers, Inc. Isolatable, water soluble, and hydrolytically stable active sulfones of poly(ethylene glycol) and related polymers for modification of surfaces and molecules
US5545529A (en) * 1993-01-27 1996-08-13 New York University Assay for detecting covalent DNA-protein complexes
US5629384A (en) * 1994-05-17 1997-05-13 Consiglio Nazionale Delle Ricerche Polymers of N-acryloylmorpholine activated at one end and conjugates with bioactive materials and surfaces
WO1998032466A1 (fr) * 1997-01-29 1998-07-30 Polymasc Pharmaceuticals Plc Procede de p.e.g.ylation
WO1998037186A1 (fr) * 1997-02-18 1998-08-27 Actinova Limited Bibliotheque d'expression in vitro de proteine ou de peptide
GB2338237A (en) * 1997-02-18 1999-12-15 Actinova Ltd In vitro peptide or protein expression library
US6552170B1 (en) 1990-04-06 2003-04-22 Amgen Inc. PEGylation reagents and compounds formed therewith
US6989147B2 (en) 1996-07-09 2006-01-24 Amgen Inc. Truncated soluble tumor necrosis factor type-I and type-II receptors

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
Biochemistry, Vol. 20, 1981 Leonard A. Zwelling et al.: "Protein-associated deoxyribonucleic acid strand breaks in L1210 cells treated with the deoxyribonucleic acid intercalating agents 4'-(9-acridinylamino)methanesulfon-m-anisidide and", page 6553-6563. *
Chem. Pharm. Bull., Vol. 36, No. 8, 1988 Paul McGoff et al.: "Analysis of Polyethylene Glycol Modified Superoxide Dismutase by Chromatographic, Electrophoretic, Light Scattering, Chemical and Enzymatic Methods ", page 3079-3091. *
The EMBO Journal, Vol. 3, No. 3, 1984 Douglas K. Trask et al.: "Rapid detection and isolation of covalent DNA/protein complexes: application to topoisomerase I and II ", page 671-676. *

Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6552170B1 (en) 1990-04-06 2003-04-22 Amgen Inc. PEGylation reagents and compounds formed therewith
US5545529A (en) * 1993-01-27 1996-08-13 New York University Assay for detecting covalent DNA-protein complexes
EP1026171A1 (fr) * 1993-08-24 2000-08-09 PolyMASC Pharmaceuticals plc Modification des polymeres
WO1995006058A1 (fr) * 1993-08-24 1995-03-02 Polymasc Pharmaceuticals Plc Modification de polymere
US5446090A (en) * 1993-11-12 1995-08-29 Shearwater Polymers, Inc. Isolatable, water soluble, and hydrolytically stable active sulfones of poly(ethylene glycol) and related polymers for modification of surfaces and molecules
US7214366B2 (en) 1993-11-12 2007-05-08 Nektar Therapeutics Al, Corporation Isolatable, water soluble, and hydrolytically stable active sulfones of poly(ethylene glycol) and related polymers for modification of surfaces and molecules
US5739208A (en) * 1993-11-12 1998-04-14 Shearwater Polymers, Inc. Isolatable, water soluble, and hydrolytically stable active sulfones of poly(ethylene glycol) and related polymers for modification of surfaces and molecules
US6894025B2 (en) 1993-11-12 2005-05-17 Nektar Therapeutics Al, Corp. Biologically active molecules having thiol moiety conjugated to polymers containing ethyl sulfone moiety
US5900461A (en) * 1993-11-12 1999-05-04 Shearwater Polymers, Inc. Isolatable, water soluble, and hydrolytically stable active sulfones of poly(ethylene glycol) and related polymers for modification of surfaces and molecules
US5631322A (en) * 1994-05-17 1997-05-20 Consiglio Nazionale Delle Ricerche Polymers of N-acryloylmorpholine activated at one end and conjugates with bioactive materials and surfaces
US5629384A (en) * 1994-05-17 1997-05-13 Consiglio Nazionale Delle Ricerche Polymers of N-acryloylmorpholine activated at one end and conjugates with bioactive materials and surfaces
US6989147B2 (en) 1996-07-09 2006-01-24 Amgen Inc. Truncated soluble tumor necrosis factor type-I and type-II receptors
US7732587B2 (en) 1996-07-09 2010-06-08 Amgen Inc. Nucleic acids encoding truncated soluble tumor necrosis factor
WO1998032466A1 (fr) * 1997-01-29 1998-07-30 Polymasc Pharmaceuticals Plc Procede de p.e.g.ylation
GB2338237A (en) * 1997-02-18 1999-12-15 Actinova Ltd In vitro peptide or protein expression library
GB2338237B (en) * 1997-02-18 2001-02-28 Actinova Ltd In vitro peptide or protein expression library
AU744791B2 (en) * 1997-02-18 2002-03-07 Isogenica Limited. In vitro peptide or protein expression library
WO1998037186A1 (fr) * 1997-02-18 1998-08-27 Actinova Limited Bibliotheque d'expression in vitro de proteine ou de peptide

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