DNA-PROTEIN CROSS LINKING

Prepared by,K.P.Senthil Kumar.,M.Sc.,M.Phil.,ADAB

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.

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.

Today, aromatic azido compounds are mostly used as photoactivatable ligand analogs.

They form highly reactive nitrenes upon irradiation because of the electron sextet in the outer electron shell of these intermediates .

In addition to the azido derivatives, photoreactive precursors forming.

Radicals or carbenes on irradiation can be used as photoaffinity labels. All of these intermediates (e.g. nitrenes), vigorously try to complete an electron octet .

To produce covalent crosslinks between proteins and DNA, various methods have been applied :

Ultraviolet (UV) irradiation, γ-irradiation, chemical methods, and even vacuum or extreme dryness.

To date, many successful attempts have been made to photocrosslink proteins to nucleic acids using different photoactivatable 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

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

2. Characterization of 8-N3dATP:Thin-layer chromatography (TLC). TLC is carried out on silica gel plates F254 or cellulose plates F. The development is performed in either isobutyric acid/water/ ammonia (66:33:1 v/v) or n-butanol/water/acetic acid (5:3:2 v/v).

Ultraviolet absorbance. Record the UV absorbance spectrum of 8-N3dATP. It shows a maximum at 280 nm. The UV absorbance of 8-N3dATP is pH dependent.

Photoreactivity. The photoreactivity of 8-N3dATP is tested by two different methods. It can either be demonstrated by the spectroscopic observation of the photolysis or by the ability of the photolabel to bind irreversibly to cellulose on thin-layer plates on UV irradiation prior to the development of the chromatogram.

3. Preparation of Azido-Modified DNAAzido-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

4. PhotocrosslinkingAn ultraviolet lamp emitting UV light at wavelengths of 300 nm and longer.1. Prepare 20–30 μL aqueous solutions containing the photoreactive 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 in the 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 after photolysis .

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.

A quantitative determination of the DNA–protein adducts is possible by densitometric measurement of the autoradiogram .

Another possibility to detect the DNA–protein adducts is the application of the nitrocellulose filter binding assay according to Braun and Merrick .

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.