UV Crosslinking of Proteins to Nucleic Acids

Lewis A. Chodosh1

1 Massachusetts Institute of Technology and Harvard Medical School, Cambridge and Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 12.5
DOI:  10.1002/0471142727.mb1205s36
Online Posting Date:  May, 2001
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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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Table of Contents

  • 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‐32P]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 CaCl 2
  • DNase I (Worthington)
  • 1 U micrococcal nuclease (Worthington)
  • 2× SDS/sample buffer (unit 10.2)
  • Fluor (Du Pont NEN Enhance or equivalent)
  • 14C‐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

Videos

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.
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