UV Crosslinking of Proteins to Nucleic Acids

互联网2013-12-31

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Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

Irradiation of protein?nucleic acid complexes with ultraviolet light causes covalent bonds to form between the nucleic acid and proteins that are in close contact with the nucleic acid. Thus, UV crosslinking may be used to selectively label DNA?binding proteins based on their specific interaction with a DNA recognition site. As a consequence of label transfer, the molecular weight of a DNA?binding protein in a crude mixture can be rapidly and reliably determined. This unit provides 3 protocols for executing DNA?protein crosslinking; one uses the halogenated thymidine analog bromodeoxyuridine (BrdU) to produce a DNA probe that is especially sensitive to UV?induced crosslinking. An alternate protocol describes crosslinking using a non?BrdU substituted probe, and another alternate protocol provides a method for in situ crosslinking.

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Basic Protocol 1: UV Crosslinking Using a Bromodeoxyuridine‐ Substituted Probe
Alternate Protocol 1: UV Crosslinking Using a Non–Bromodeoxyuridine‐Substituted Probe
Alternate Protocol 2: UV Crosslinking In Situ
Reagents and Solutions
Commentary
Figures

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Materials

Basic Protocol 1: UV Crosslinking Using a Bromodeoxyuridine‐ Substituted Probe

Materials

Single‐stranded M13 vector with desired binding site (unit 1.15 )
17‐bp M13 universal primer (Pharmacia)
1× and 10× restriction endonuclease buffer (50 mM NaCl and 500 mM NaCl, respectively; unit 3.1 )
3000 Ci/mmol [α‐³² P]dCTP (unit 3.4 )
recipe50× dNTP/BrdU solution
0.1 M dithiothreitol (DTT)
25 U Klenow fragment of E. coli DNA polymerase I (unit 3.5 )
Ammonium acetate
100% ethanol
TE buffer ( appendix 22 )
Buffered extract containing DNA‐binding protein
Bulk carrier DNA, e.g., poly(dI‐dC)⋅poly(dI‐dC)
0.5 M CaCl₂
DNase I (Worthington)
1 U micrococcal nuclease (Worthington)
2× SDS/sample buffer (unit 10.2 )
Fluor (Du Pont NEN Enhance or equivalent)
¹⁴ C‐labeled protein markers (unit 10.2 )

DEAE membrane (Schleicher & Schuell NA45)
1.5‐ml round‐bottom screw‐cap vial (Nunc or equivalent)
16°, 37°, 68°, 90°, and 100°C water baths
UV transilluminator (305 nm, 7000 µW/cm² ; Fotodyne)

Additional reagents and equipment for digesting with restriction nucleases (unit 3.1 ), labeling with Klenow fragment (unit 3.5 ), agarose gel electrophoresis (unit 2.5 ), mobility shift DNA‐binding assay (unit 12.2 ), ethidium bromide dot quantitation (unit 2.6 ), SDS‐polyacrylamide gel electrophoresis (units 10.2 and 10.3 ), and autoradiography ( 3.NaN )

Alternate Protocol 1: UV Crosslinking Using a Non–Bromodeoxyuridine‐Substituted Probe

Additional Materials

recipe50× dNTP/TTP solution
UV transilluminator (254 nm)

Alternate Protocol 2: UV Crosslinking In Situ

Additional Materials

Low gelling/melting temperature agarose (unit 2.6 )
1× TBE buffer ( appendix 22 )
SDS gel solution

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Figures

Figure 12.5.1 Experimental setup for UV crosslinking DNA‐binding proteins to uniformly labeled DNA.

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Figure 12.5.2 Representation of autoradiogram of a typical UV crosslinking experiment. As described in commentary, procedure involves UV crosslinking of protein to DNA probe, digestion of free DNA with DNase, and electrophoresis by SDS‐PAGE of the treated binding reactions. Molecular weights of ¹⁴ C‐labeled protein markers in Lane 1 are indicated to the left. Lanes 2 through 10 each contained a ¹⁴ C‐labeled DNA probe with a site for a binding protein X. Samples applied to the lanes differed as follows (see also anticipated results): Lane 2 —no protein; Lane 3— no UV irradiation; Lane 4 —5 min irradiation of complete binding reaction; Lane 5 —15 min irradiation of complete binding reaction; Lane 6 —30 min irradiation of complete binding reaction; Lane 7 —60 min irradiation of complete binding reaction; Lane 8 —60 min irradiation of complete binding reaction then proteinase K treatment; Lane 9 —60 min irradiation of complete binding reaction plus 100‐fold molar excess of unlabeled probe containing a binding site for protein X; Lane 10 —60 min irradiation of complete binding reaction plus 100‐fold molar excess of unlabeled mutant probe lacking a binding site for protein X.

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Literature Cited

Literature Cited
	Chodosh, L.A., Carthew, R.W., and Sharp, P.A. 1986. A single polypeptide possesses the binding and transcription activities of the adenovirus major late transcription factor. Mol. Cell. Biol. 6:4723‐4733.
	Hillel, Z. and Wu, C.‐W. 1978. Photochemical cross‐linking studies on the interaction of Escherichia coli RNA polymerase with T7 DNA. Biochemistry 17:2954‐2961.
	Lin, S.‐Y. and Riggs, A.D. 1974. Photochemical attachment of lac repressor to bromodeoxyuridine‐substituted lac operator by ultraviolet radiation. Proc. Natl. Acad. Sci. U.S.A. 71:947‐951.
	Markowitz, A. 1972. Ultraviolet light–induced stable complexes of DNA and DNA polymerase. Biochim. Biophys. Acta 281:522‐534.
	Simpson, R.B. 1979. The molecular topography of RNA polymerase–promoter interaction. Cell 18:277‐285.
	Wu, C., Wilson, S., Walker, B., Dawid, I., Paisley, T., Zimarino, V., and Ueda, H. 1987. Purification and properties of Drosophila heat shock activator protein. Science 238:1247‐1253.
Key References
	Chodosh et al., 1986. See above.
	Describes UV crosslinking in crude mammalian extracts and demonstrates specificity of binding of the crosslinked proteins. Compares the technique with other methods for determining the molecular weight of DNA‐binding proteins.
	Hillel and Wu, 1978. See above.
	An excellent study of the differences in polymerase‐DNA contacts in specific and nonspecific complexes.
	Lin and Riggs, 1974. See above.
	A seminal paper describing the use of BrdU in photochemical crosslinking.