Summary
Several methods have been developed to site-specifically incorporate photoreactive nucleotide analogs into DNA for the purpose of identifying the proteins and their domains that are in contact with particular regions of DNA. The synthesis of several deoxynucleotide analogs that have a photoreactive group tethered to the nucleotide base and the incorporation of these analogs into DNA are described. In a second approach, oligonucleotide with a photoreactive group attached to the phosphate backbone is chemically synthesized. The photoreactive oligonucleotide is then enzymatically incorporated into DNA by annealing it to a complementary DNA template and extending with DNA polymerase. Both approaches have been effectively used to map protein–DNA interactions in large multisubunit complexes such as the eukaryotic transcription or ATP-dependent chromatin remodeling complexes. Not only do these techniques map the binding sites of the various subunits in these complexes, but when coupled with peptide mapping also determine the protein domain that is in close proximity to the different DNA sites. The strength of these techniques is the ability to scan a large number of potential sites by making combinations of different DNA probes and is facilitated by using an immobilized DNA template for synthesis.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Geiduschek, E. P., and Kassavetis, G. A. (2001). The RNA polymerase III transcription apparatus, J Mol Biol 310, 1–26.
Kassavetis, G. A., and Geiduschek, E. P. (2006). Transcription factor TFIIIB and transcription by RNA polymerase III, Biochem Soc Trans 34, 1082–1087.
Persinger, J., and Bartholomew, B. (2001). Site-directed DNA photoaffinity labeling of RNA polymerase III transcription complexes, Methods Mol Biol 148, 363–381.
Gangaraju, V. K., and Bartholomew, B. (2007). Mechanisms of ATP dependent chromatin remodeling, Mutat Res 618, 3–17.
Dang, W., and Bartholomew, B. (2007). Domain architecture of the catalytic subunit in the ISW2-nucleosome complex, Mol Cell Biol 27, 8306–8317.
Bartholomew, B., Kassavetis, G. A., Braun, B. R., and Geiduschek, E. P. (1990). The subunit structure of Saccharomyces cerevisiae transcription factor IIIC probed with a novel photocrosslinking reagent, Embo J 9, 2197–2205.
Lannutti, B. J., Persinger, J., and Bartholomew, B. (1996). Probing the protein–DNA contacts of a yeast RNA polymerase III transcription complex in a crude extract: solid phase synthesis of DNA photoaffinity probes containing a novel photoreactive deoxycytidine analog, Biochemistry 35, 9821–9831.
Zofall, M., and Bartholomew, B. (2000). Two novel dATP analogs for DNA photoaffinity labeling, Nucleic Acids Res 28, 4382–4390.
Tate, J. J., Persinger, J., and Bartholomew, B. (1998). Survey of four different photoreactive moieties for DNA photoaffinity labeling of yeast RNA polymerase III transcription complexes, Nucleic Acids Res 26, 1421–1426.
Bartholomew, B., Braun, B. R., Kassavetis, G. A., and Geiduschek, E. P. (1994). Probing close DNA contacts of RNA polymerase III transcription complexes with the photoactive nucleoside 4-thiodeoxythymidine, J Biol Chem 269, 18090–18095.
Persinger, J., and Bartholomew, B. (1996). Mapping the contacts of yeast TFIIIB and RNA polymerase III at various distances from the major groove of DNA by DNA photoaffinity labeling, J Biol Chem 271, 33039–33046.
Galardy, R. E., Craig, L. C., Jamieson, J. D., and Printz, M. P. (1974). Photoaffinity label-ing of peptide hormone binding sites, J Biol Chem 249, 3510–3518.
Burgermeister, W., Nassal, M., Wieland, T., and Helmreich, E. J. (1983). A carbene-generating photoaffinity probe for beta-adrenergic receptors, Biochim Biophys Acta 729, 219–228.
Persinger, J., Sengupta, S. M., and Bartholomew, B. (1999). Spatial organization of the core region of yeast TFIIIB-DNA complexes, Mol Cell Biol 19, 5218–5234.
Kagalwala, M. N., Glaus, B. J., Dang, W., Zofall, M., and Bartholomew, B. (2004). Topography of the ISW2-nucleosome complex: insights into nucleosome spacing and chromatin remodeling, Embo J 23, 2092–2104.
Sengupta, S. M., Persinger, J., Bartholomew, B., and Peterson, C. L. (1999). Use of DNA photoaffinity labeling to study nucleosome remodeling by SWI/SNF, Methods 19, 434–446.
Sengupta, S. M., VanKanegan, M., Persinger, J., Logie, C., Cairns, B. R., Peterson, C. L., and Bartholomew, B. (2001). The interactions of yeast SWI/SNF and RSC with the nucleosome before and after chromatin remodeling, J Biol Chem 276, 12636–12644.
Bartholomew, B., Tinker, R. L., Kassavetis, G. A., and Geiduschek, E. P. (1995). Photochemical cross-linking assay for DNA tracking by replication proteins, Methods Enzymol 262, 476–494.
Bartholomew, B., Durkovich, D., Kassavetis, G. A., and Geiduschek, E. P. (1993). Orientation and topography of RNA polymerase III in transcription complexes, Mol Cell Biol 13, 942–952.
Gallagher, S. R. (2007). One-dimensional SDS gel electrophoresis of proteins, Curr Protoc Cell Biol Chapter 6, Unit 6 1.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Persinger, J., Bartholomew, B. (2009). Site-Directed DNA Crosslinking of Large Multisubunit Protein-DNA Complexes. In: Leblanc, B., Moss, T. (eds) DNA-Protein Interactions. Methods in Molecular Biology™, vol 543. Humana Press. https://doi.org/10.1007/978-1-60327-015-1_27
Download citation
DOI: https://doi.org/10.1007/978-1-60327-015-1_27
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-60327-014-4
Online ISBN: 978-1-60327-015-1
eBook Packages: Springer Protocols