Dna prt cross linking


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Unit II Paper 10 MBT II for Msc BT

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Dna prt cross linking

  1. 1. Prepared by,K.P.Senthil Kumar.,M.Sc.,M.Phil.,ADAB
  2. 2. In biological systems,  Photoreactive derivatives to study specific interactions  Ex:Receptor molecules with their ligands by photoaffinity labeling  Receptors are generally proteins e.g., enzymes, immunoglobulins, or hormone receptors. The ligands differ widely in their molecular structure e.g., sugars, amino acids, nucleotides, or oligomers of these compound.
  3. 3. The advantage  Its Preferred over affinity labeling, or chemical modification with group-specific reagents  Photoactivatable nonreactive precursors can be activated at will by irradiation . These reagents do not bind covalently to the protein unless activated.  On irradiation of the precursors, highly reactive intermediates are formed.  That react indiscriminately with all surrounding groups.  After activation, a photoaffinity label, interacting at the specific binding site, can label all the different amino acid residues of the binding area.
  4. 4.  Today, aromatic azido compounds are mostly usedas photoactivatable ligand analogs. They form highly reactive nitrenes upon irradiationbecause of the electron sextet in the outer electronshell of these intermediates . In addition to the azido derivatives, photoreactiveprecursors forming. Radicals or carbenes on irradiation can be used asphotoaffinity labels. All of these intermediates (e.g.nitrenes), vigorously try to complete an electron octet.
  5. 5. To produce covalent crosslinks between proteins andDNA, various methods have been applied :  Ultraviolet (UV) irradiation, γ-irradiation, chemical methods, and even vacuum or extreme dryness.
  6. 6. To date, many successful attempts have been made tophotocrosslink proteins to nucleic acids using differentphotoactivatable deoxynucleotides.  5-bromo-,5-iodo-, 5-azido-, and 5-[N-(p-azidobenzoyl)-3-aminoallyl]-2-deoxyuridine- 5-monophosphate , 4-thio-2-deoxythymidine-5-monophosphate , and 8-azido-2-deoxyadenosine-5-monophosphate (have been incorporated into deoxyribonucleic acids to bind DNA covalently to adjacent proteins
  7. 7. Methods1. Synthesis of 8-N3dATP: The synthesis of 8-N3dATP is performed principally by analogy to the synthesis of 8-N3ATP . In the first step, bromine exchanges the hydrogen at position 8 of the adenine ring. Then, the bromine is substituted by the azido group
  8. 8. 2. Characterization of 8-N3dATP:Thin-layer chromatography (TLC). TLC is carried out onsilica gel plates F254 or cellulose plates F. The development isperformed in either isobutyric acid/water/ ammonia (66:33:1v/v) or n-butanol/water/acetic acid (5:3:2 v/v).Ultraviolet absorbance. Record the UV absorbance spectrumof 8-N3dATP. It shows a maximum at 280 nm. The UVabsorbance of 8-N3dATP is pH dependent.
  9. 9. Photoreactivity. The photoreactivity of 8-N3dATP is testedby two different methods. It can either be demonstrated by thespectroscopic observation of the photolysis or by the ability ofthe photolabel to bind irreversibly to cellulose on thin-layerplates on UV irradiation prior to the development of thechromatogram.
  10. 10. 3. Preparation of Azido-Modified DNA Azido-modified and [32P]-labeled DNA are prepared by nick translation.  The detailed and exact composition of the reaction medium depends strongly on the size as well as on the amount of the DNA to be modified. The optimal ratio of DNA, DNase I, and DNA polymerase I (Kornberg enzyme) should be tested in preliminary experiments
  11. 11. 4. PhotocrosslinkingAn ultraviolet lamp emitting UV light at wavelengthsof 300 nm and longer.1. Prepare 20–30 μL aqueous solutions containing thephotoreactive DNA (0.5 pmol)and the protein (1–25 pmol) to be cross-linked .2. Incubate the reaction mixture for 10 min at 37 C inthe dark.3. Expose the sample to UV irradiation . The irradiation times can be chosen in a range from 1 s to 60 min .4. Keep the solutions in the dark before and afterphotolysis .
  12. 12. 5. Analysis of DNA-Protein Adducts  By polyacrylamide gel electrophoresis of the irradiated samples followed by autoradiography. SDS–polyacrylamide gel electrophoresis should be performed immediately after photocrosslinking according to Laemmli with some variations. After the addition of 20 mg/mL of bromophenol blue, the samples are loaded onto a SDS– polyacrylamide gel of 5% polyacrylamide (separating gel) with an overlay of 3.5% polyacrylamide (stacking gel) containing 1% SDS. After the electrophoretic separation, the gels are silver-stained according to Adams and Sammons ,dried, and exposed to X-ray film at –70 C.
  13. 13. A quantitative determination of the DNA–protein adducts is possible by densitometric measurement of the autoradiogram .Another possibility to detect the DNA–protein adducts isthe application of the nitrocellulose filter binding assayaccording to Braun and Merrick .
  14. 14. Reference: •Rainer Meffert, Klaus Dose, Gabriele Rathgeber, and Hans-Jochen Schäfer. Ultraviolet Crosslinking of DNA– Protein Complexes via 8-Azidoadenine. Chapter 23. Methods in molecular biology. 2001(43) 323-335; Dna – Protein Interactions Principles and protocols. Humana Press Inc.