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Crystallization of Protein-DNA Complexes

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Macromolecular Crystallography Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 363))

Abstract

Determining the crystal structure of a protein-DNA complex can provide a wealth of information regarding protein function and mechanism. The foundation for all successful X-ray structure determination is the ability to produce diffraction-quality crystals. Crystallization of protein-DNA complexes often presents unique challenges because of the additional parameters involved. This chapter will outline many of those challenges, including choice of DNA and formation of a stable protein-DNA complex and provide guidance in preparing for crystallization experiments. Additionally, techniques for oligonucleotide purification, sample preparation, and crystallization methods are provided. Careful thought and initial analysis of the protein-DNA complex prior to crystallization experiments followed by optimization of crystallization parameters can greatly increase the likelihood of producing well-diffracting crystals.

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References

  1. Ducruix, A. and R. Giegé, R. (1999) Crystallization of Nucleic Acids and Proteins. A Practical Approach, ed. Oxford University Press, Oxford, UK, pp. 121–238.

    Google Scholar 

  2. Nucleic Acid Database, Rutgers University. (http://ndbserver.rutgers.edu) Accessed June, 2006.

  3. Scopes, R. K. (1987) Protein Purification Principles and Practice, 2nd ed. Springer-Verlag, New York, NY.

    Google Scholar 

  4. Deutscher, M. P. (1990) Guide to Protein Purification. Methods in Enzymology, vol. 182. Academic Press, New York, NY.

    Google Scholar 

  5. Ausubel, F. M. (1992) Short Protocols in Molecular Biology, 2nd ed. Greene Publishing Assoc. and John Wiley and Sons, New York, NY.

    Google Scholar 

  6. Beese, L. and Steitz, T. (1991) Structural basis for the 3′-5′ exonuclease activity of Escherichia coli DNA polymerase I: a two metal ion mechanism. EMBO J. 10, 25–33.

    CAS  PubMed  Google Scholar 

  7. Hollis, T. and Ellenberger, T. (2000) DNA bending and a flip-out mechanism for base excision by the helix-hairpin-helix DNA glycosylase, Escherichia coli AlkA. EMBO J. 19, 758–766.

    Article  CAS  PubMed  Google Scholar 

  8. Lau, A., Scharer, O. D., Samson, L., Verdine, G. L., and Ellenberger, T. (1998) Crystal structure of a human alkylbase-DNA repair enzyme complexed to DNA: mechanisms for nucleotide flipping and base excision. Cell 95, 249–258.

    Article  CAS  PubMed  Google Scholar 

  9. Davies, D. R., Interthal, H., Champoux, J. J., and Hol, W. G. (2003) Crystal structure of a transition state mimic for tdp1 assembled from vanadate, DNA, and a topoisomerase I-derived peptide. Chem. Biol. 10, 139–147.

    Article  CAS  PubMed  Google Scholar 

  10. Doublié, S., Tabor, S., Long, A. M., Richardson, C. C., and Ellenberger, T. (1998) Crystal structure of a bacteriophage T7 DNA replication complex at 2.2 Å resolution. Nature 391, 251–258.

    Article  PubMed  Google Scholar 

  11. Fromme, J. and Verdine, G. (2003) Structure of a trapped endonuclease III-DNA covalent intermediate. EMBO J. 22, 3461–3471.

    Article  CAS  PubMed  Google Scholar 

  12. Redinbo, M. R., Stewart, L., Kuhn, P., Champoux, J. J., and Hol, W. G. (1998) Crystal structures of human topoisomerase I in covalent and noncovalent complexes with DNA. Science 279, 1504–1513.

    Article  CAS  PubMed  Google Scholar 

  13. He, C. and Verdine, G. (2002) Trapping distinct structural states of a protein/DNA interaction through disulfide crosslinking. Chem. Biol. 9, 1297–1303.

    Article  CAS  PubMed  Google Scholar 

  14. Huang, H., S. Harrison, and G. Verdine. (2000) Trapping of a catalytic HIV reverse transcriptase-template:primer complex through a disulfide bond. Chem. Biol. 7, 355–364.

    Article  CAS  PubMed  Google Scholar 

  15. Sarafianos, S., Clark, A. D., Jr, Tuske, S., et al. (2003) Trapping HIV-1 reverse transcriptase before and after translocation on DNA. J. Biol. Chem. 278, 16,280–16,288.

    Article  CAS  PubMed  Google Scholar 

  16. Brautigam, C., Aschheim, K., and Steitz, T. (1999) Structural elucidation of the binding and inhibitory properties of lanthanide (III) ions at the 3′-5′ exonucleolytic active site of the Klenow fragment. Chem. Biol. 6, 901–908.

    Article  CAS  PubMed  Google Scholar 

  17. Brautigam, C. and Steitz, T. (1998) Structural principles for the inhibition of the 3′-5′ exonuclease activity of Escherichia coli DNA polymerase I by phosphorothioates. J. Mol. Biol. 277, 363–377.

    Article  CAS  PubMed  Google Scholar 

  18. McPherson, A. (1999) Crystallization of Biological Macromolecules. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

    Google Scholar 

  19. Carter, C. W. J. and Carter, C. W. (1979) Protein crystallization using incomplete factorial experiments. J. Biol. Chem. 254, 12,219–12,223.

    CAS  PubMed  Google Scholar 

  20. Betts, L. et al. (1989) Incomplete factorial search for conditions leading to high quality crystals of Escherichia coli cytidine deaminase complexed to a transition state analog inhibitor. J. Biol. Chem. 264, 6737–6740.

    CAS  PubMed  Google Scholar 

  21. Ellenberger, T. and Doublié, S. (1996) Use of perfusion chromatography technology for the rapid purification of synthetic oligonucleotides used in DNA/DNA-binding protein structural studies. Biochemica 1, 12–13.

    Google Scholar 

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Hollis, T. (2007). Crystallization of Protein-DNA Complexes. In: Walker, J.M., Doublié, S. (eds) Macromolecular Crystallography Protocols. Methods in Molecular Biology, vol 363. Humana Press. https://doi.org/10.1007/978-1-59745-209-0_11

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  • DOI: https://doi.org/10.1007/978-1-59745-209-0_11

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-292-6

  • Online ISBN: 978-1-59745-209-0

  • eBook Packages: Springer Protocols

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