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SSB Binding to ssDNA Using Isothermal Titration Calorimetry

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Single-Stranded DNA Binding Proteins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 922))

Abstract

Isothermal titration calorimetry (ITC) is a powerful method for studying protein–DNA interactions in solution. As long as binding is accompanied by an appreciable enthalpy change, ITC studies can yield quantitative information on stoichiometries, binding energetics (affinity, binding enthalpy and entropy) and potential site–site interactions (cooperativity). This can provide a full thermodynamic description of an interacting system which is necessary to understand the stability and specificity of protein–DNA interactions and to correlate the activities or functions of different species. Here we describe procedures to perform and analyze ITC studies using as examples, the E. coli SSB (homotetramer with 4 OB-folds) and D. radiodurans SSB (homodimer with 4 OB-folds). For oligomeric protein systems such as these, we emphasize the need to be aware of the likelihood that solution conditions will influence not only the affinity and enthalpy of binding but also the mode by which the SSB oligomer binds ssDNA.

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References

  1. Wyman J, Gill SJ (1990) Bindng and linkage. University Science Books, Mill Valley, CA

    Google Scholar 

  2. Lohman TM, Overman LB, Ferrari ME, Kozlov AG (1996) A highly salt-dependent enthalpy change for Escherichia coli SSB protein-nucleic acid binding due to ion–protein interactions. Biochemistry 35:5272–5279

    Article  PubMed  CAS  Google Scholar 

  3. Kozlov AG, Lohman TM (1998) Calorimetric studies of E-coli SSB protein single-stranded DNA interactions. Effects of monovalent salts on binding enthalpy. J Mol Biol 278:999–1014

    Article  PubMed  CAS  Google Scholar 

  4. Kozlov AG, Lohman TM (1999) Adenine base unstacking dominates the observed enthalpy and heat capacity changes for the Escherichia coli SSB tetramer binding to single-stranded oligoadenylates. Biochemistry 38:7388–7397

    Article  PubMed  CAS  Google Scholar 

  5. Kozlov AG, Lohman TM (2000) Large contributions of coupled protonation equilibria to the observed enthalpy and heat capacity changes for ssDNA binding to Escherichia coli SSB protein. Proteins Suppl 4:8–22

    Article  Google Scholar 

  6. Kozlov AG, Lohman TM (2006) Effects of monovalent anions on a temperature-dependent heat capacity change for Escherichia coli SSB tetramer binding to single-stranded DNA. Biochemistry 45:5190–5205

    Article  PubMed  CAS  Google Scholar 

  7. Kozlov AG, Lohman TM (2011) E. coli SSB tetramer binds the first and second molecules of (dT)(35) with heat capacities of opposite sign. Biophys Chem 159:48–57

    Article  PubMed  CAS  Google Scholar 

  8. Kozlov AG, Eggington JM, Cox MM, Lohman TM (2010) Binding of the dimeric Deinococcus radiodurans single-stranded DNA binding protein to single-stranded DNA. Biochemistry 49:8266–8275

    Article  PubMed  CAS  Google Scholar 

  9. Kumaran S, Kozlov AG, Lohman TM (2006) Saccharomyces cerevisiae replication protein A binds to single-stranded DNA in multiple salt-dependent modes. Biochemistry 45:11958–11973

    Article  PubMed  CAS  Google Scholar 

  10. Lohman TM, Ferrari ME (1994) Escherichia coli single-stranded DNA-binding protein: multiple DNA-binding modes and cooperativities. Annu Rev Biochem 63:527–570

    Article  PubMed  CAS  Google Scholar 

  11. Raghunathan S, Ricard CS, Lohman TM, Waksman G (1997) Crystal structure of the homo-tetrameric DNA binding domain of Escherichia coli single-stranded DNA-binding protein determined by multiwavelength X-ray diffraction on the selenomethionyl protein at 2.9-A resolution. Proc Natl Acad Sci U S A 94:6652–6657

    Article  PubMed  CAS  Google Scholar 

  12. Bujalowski W, Overman LB, Lohman TM (1988) Binding mode transitions of Escherichia coli single strand binding protein-single-stranded DNA complexes. Cation, anion, pH, and binding density effects. J Biol Chem 263:4629–4640

    PubMed  CAS  Google Scholar 

  13. Savvides SN, Raghunathan S, Futterer K, Kozlov AG, Lohman TM, Waksman G (2004) The C-terminal domain of full-length E-coli SSB is disordered even when bound to DNA. Protein Sci 13:1942–1947

    Article  PubMed  CAS  Google Scholar 

  14. Shereda RD, Kozlov AG, Lohman TM, Cox MM, Keck JL (2008) SSB as an organizer/mobilizer of genome maintenance complexes. Crit Rev Biochem Mol Biol 43:289–318

    Article  PubMed  CAS  Google Scholar 

  15. Overman LB, Lohman TM (1994) Linkage of pH, anion and cation effects in protein-nucleic acid equilibria. Escherichia coli SSB protein-single stranded nucleic acid interactions. J Mol Biol 236:165–178

    Article  PubMed  CAS  Google Scholar 

  16. Bernstein DA, Eggington JM, Killoran MP, Misic AM, Cox MM, Keck JL (2004) Crystal structure of the Deinococcus radiodurans single-stranded DNA-binding protein suggests a mechanism for coping with DNA damage. Proc Natl Acad Sci U S A 101:8575–8580

    Article  PubMed  CAS  Google Scholar 

  17. Lohman TM, Green JM, Beyer RS (1986) Large-scale overproduction and rapid purification of the Escherichia coli ssb gene product. Expression of the ssb gene under lambda PL control. Biochemistry 25:21–25

    Article  PubMed  CAS  Google Scholar 

  18. Eggington JM, Haruta N, Wood EA, Cox MM (2004) The single-stranded DNA-binding protein of Deinococcus radiodurans. BMC Microbiol 4:2

    Article  PubMed  Google Scholar 

  19. Ferrari ME, Bujalowski W, Lohman TM (1994) Co-operative binding of Escherichia coli SSB tetramers to single-stranded DNA in the (SSB)35 binding mode. J Mol Biol 236:106–123

    Article  PubMed  CAS  Google Scholar 

  20. Wong I, Chao KL, Bujalowski W, Lohman TM (1992) DNA-induced dimerization of the Escherichia coli rep helicase, Allosteric effects of single-stranded and duplex DNA. J Biol Chem 267:7596–7610

    PubMed  CAS  Google Scholar 

  21. Wiseman T, Williston S, Brandts JF, Lin LN (1989) Rapid measurement of binding constants and heats of binding using a new titration calorimeter. Anal Biochem 179:131–137

    Article  PubMed  CAS  Google Scholar 

  22. Bujalowski W, Lohman TM (1989) Negative co-operativity in Escherichia coli single strand binding protein–oligonucleotide interactions. I. Evidence and a quantitative model. J Mol Biol 207:249–268

    Article  PubMed  CAS  Google Scholar 

  23. Weast RC (ed) (1974–1975) Handbook of chemistry and physics, 55 edn. CRC Press.

    Google Scholar 

  24. Lohman TM, Chao K, Green JM, Sage S, Runyon GT (1989) Large-scale purification and characterization of the Escherichia coli rep gene product. J Biol Chem 264:10139–10147

    PubMed  CAS  Google Scholar 

  25. Cantor CR, Warshaw MM, Shapiro H (1970) Oligonucleotide interactions. 3. Circular dichroism studies of the conformation of deoxyoligonucleotides. Biopolymers 9:1059–1077

    Article  PubMed  CAS  Google Scholar 

  26. Fasman GD (ed) (1975) Handbook of biochemistry and molecular biology, vol 1: nucleic acids, 3rd edn., CRC Press.

    Google Scholar 

  27. Holbrook JA, Capp MW, Saecker RM, Record MT Jr (1999) Enthalpy and heat capacity changes for formation of an oligomeric DNA duplex: interpretation in terms of coupled processes of formation and association of single-stranded helices. Biochemistry 38:8409–8422

    Article  PubMed  CAS  Google Scholar 

  28. Lohman TM, Bujalowski W (1991) Thermodynamic methods for model-independent determination of equilibrium binding isotherms for protein–DNA interactions: spectroscopic approaches to monitor binding. Methods Enzymol 208:258–290

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

We thank Dr. Edwin Antony and Dr. Binh Nguyen for careful reading of the manuscript and useful suggestions. This work was supported in part by grants to T.M.L. from NIH (GM030498 and GM045948).

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO, USA

    Alexander G. Kozlov & Timothy M. Lohman

Authors
  1. Alexander G. Kozlov
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  2. Timothy M. Lohman
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Corresponding author

Correspondence to Timothy M. Lohman .

Editor information

Editors and Affiliations

  1. School of Medicine and Public Health, Department of Biomolecular Chemistry, University of Wisconsin, Henry Mall 420, Madison, 53706, USA

    James L. Keck

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Kozlov, A.G., Lohman, T.M. (2012). SSB Binding to ssDNA Using Isothermal Titration Calorimetry. In: Keck, J. (eds) Single-Stranded DNA Binding Proteins. Methods in Molecular Biology, vol 922. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-032-8_3

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  • DOI: https://doi.org/10.1007/978-1-62703-032-8_3

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-031-1

  • Online ISBN: 978-1-62703-032-8

  • eBook Packages: Springer Protocols

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