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Metal ion and DNA binding by single-chain PvuII endonuclease: lessons from the linker

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Abstract

Understanding the roles of metal ions in restriction enzymes has been complicated by both the presence of two metal ions in many active sites and their homodimeric structure. Using a single-chain form of the wild-type restriction enzyme PvuII (scWT) in which subunits are fused with a short polypeptide linker (Simoncsits et al. in J. Mol. Biol. 309:89–97, 2001), we have characterized metal ion and DNA binding behavior in one subunit and examined the effects of the linker on dimer behavior. scWT exhibits heteronuclear single quantum coherence NMR spectra similar to those of native wild-type PvuII (WT). For scWT, isothermal titration calorimetry data fit to two Ca(II) sites per subunit with low-millimolar K ds. The variant scWT|E68A, in which metal ion binding in one subunit is abolished by mutation, also binds two Ca(II) ions in the WT subunit with low-millimolar K ds. When there are no added metal ions, DNA binding affinity for scWT is tenfold stronger than that of the native WT, but tenfold weaker at saturating Ca(II) concentration. In the presence of Ca(II), scWT|E68A binds target DNA similarly to scWT, indicating that high-affinity substrate binding can be carried energetically by one metal-ion-binding subunit. Global analysis of DNA binding data for scWT|E68A suggests that the metal-ion-dependent behaviors observed for WT are reflective of independent subunit behavior. This characterization provides an understanding of subunit contributions in a homodimeric context.

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Abbreviations

HEX:

Hexachlorofluorescein

HSQC:

Heteronuclear single quantum coherence

ITC:

Isothermal titration calorimetry

scWT:

Single-chain wild-type PvuII endonuclease

WT:

Wild-type PvuII endonuclease

References

  1. Pingoud A (ed) (2004) Nucleic acids and molecular biology, vol 14. Springer, Berlin

  2. Dupureur CM (2008) Curr Chem Biol 2:159–173

    Article  Google Scholar 

  3. Dupureur CM (2010) Metallomics 2:609–620

    Article  PubMed  CAS  Google Scholar 

  4. Wende W, Stahl F, Pingoud A (1996) Biol Chem 377:625–632

    PubMed  CAS  Google Scholar 

  5. Stahl F, Wende W, Jeltsch A, Pingoud A (1996) Proc Natl Acad Sci USA 93:6175–6180

    Article  PubMed  CAS  Google Scholar 

  6. Simoncsits A, Tjornhammar M-L, Rasko T, Kiss A, Pongor S (2001) J Mol Biol 309:89–97

    Article  PubMed  CAS  Google Scholar 

  7. Kobayashi I (2004) In: Pingoud A (ed) Nucleic acids and molecular biology, vol 14. Springer, Berlin, pp 19–62

  8. Xie F, Qureshi S, Papadakos GA, Dupureur CM (2008) Biochemistry 47:12540

    Article  PubMed  CAS  Google Scholar 

  9. Xie F, Dupureur CM (2009) Arch Biochem Biophys 483:1–9

    Article  PubMed  CAS  Google Scholar 

  10. Prasannan CB, Xie F, Dupureur CM (2010) J Biol Inorg Chem 15:533–545

    Article  PubMed  CAS  Google Scholar 

  11. Dupureur CM (2005) Biochemistry 45:5065–5074

    Article  Google Scholar 

  12. Simoncsits A, Chen J, Percipalle P, Wang S, Toro I, Pongor S (1997) J Mol Biol 267:118–131

    Article  PubMed  CAS  Google Scholar 

  13. Balendiran K, Bonventre J, Knott R, Jack W, Benner J, Schildkraut I, Anderson JE (1994) Proteins Struct Funct Genet 19:77–79

    Article  PubMed  CAS  Google Scholar 

  14. Dupureur CM, Hallman LM (1999) Eur J Biochem 261:261–268

    Article  PubMed  CAS  Google Scholar 

  15. Bowen LM, Muller G, Riehl JP, Dupureur CM (2004) Biochemistry 43:15286–15295

    Article  PubMed  CAS  Google Scholar 

  16. Papadakos GA, Nastri H, Riggs P, Dupureur CM (2007) J Biol Inorg Chem 12:557–569

    Article  PubMed  CAS  Google Scholar 

  17. Bowen LM, Dupureur CM (2003) Biochemistry 42:12643–12653

    Article  PubMed  CAS  Google Scholar 

  18. Kuzmic P (1996) Anal Biochem 237:260–273

    Article  PubMed  CAS  Google Scholar 

  19. José TJ, Conlan LH, Dupureur CM (1999) J Biol Inorg Chem 4:814–823

    Article  PubMed  Google Scholar 

  20. Dupureur CM, Conlan LH (2000) Biochemistry 39:10921–10927

    Article  PubMed  CAS  Google Scholar 

  21. Conlan LH, Dupureur CM (2002) Biochemistry 41:1335–1342

    Article  PubMed  CAS  Google Scholar 

  22. Conlan LH, Dupureur CM (2002) Biochemistry 41:14848–14855

    Article  PubMed  CAS  Google Scholar 

  23. Dupureur CM, Dominguez JMA (2001) Biochemistry 40:387–394

    Google Scholar 

  24. Hadden JM, Declais AC, Phillips SE, Lilley DM (2002) EMBO J 21:3505–3515

    Article  PubMed  CAS  Google Scholar 

  25. King J, Bowen L, Dupureur CM (2004) Biochemistry 43:8551–8559

    Article  PubMed  CAS  Google Scholar 

  26. Beechem JM (1992) Methods Enzymol 210:37–54

    Article  PubMed  CAS  Google Scholar 

  27. Feng M, Patel D, Dervan JJ, Ceska T, Suck D, Haq I, Sayers JR (2004) Nat Struct Mol Biol 11:450–456

    Article  PubMed  CAS  Google Scholar 

  28. Syson K, Tomlinson C, Chapados BR, Sayers JR, Tainer JA, Williams NH, Grasby JA (2008) J Biol Chem 283:28741–28746

    Article  PubMed  CAS  Google Scholar 

  29. Zheng L, Li M, Shan J, Krishnamoorthi R, Shen B (2002) Biochemistry 41:10323–10331

    Article  PubMed  CAS  Google Scholar 

  30. Kurpiewski MR, Engler LE, Wozniak LA, Kobylanska A, Koziolkiewicz M, Stec WJ, Jen-Jacobson L (2004) Structure 12:1775–1788

    Article  PubMed  CAS  Google Scholar 

  31. Chen DF, Liu QA, Chen XW, Zhao XL, Chen YW (1991) Nucleic Acids Res 19:5703–5705

    Article  PubMed  CAS  Google Scholar 

  32. Cheng X, Balendiran K, Schildkraut I, Anderson JE (1994) EMBO J 13:3927–3935

    PubMed  CAS  Google Scholar 

  33. Mei G, Di Venere A, Rosato N, Finazzi-Agro A (2005) FEBS J 272:16–27

    Article  PubMed  CAS  Google Scholar 

  34. Giedroc DP, Arunkumar AI (2007) Dalton Trans 3107–3120

  35. Grossoehme NE, Giedroc DP (2009) J Am Chem Soc 131:17860–17870

    Article  PubMed  CAS  Google Scholar 

  36. Wah DA, Hirsch JA, Dorner LF, Schildkraut I, Aggarwal AK (1997) Nature 388:97–100

    Article  PubMed  CAS  Google Scholar 

  37. Horton JR, Nastri HG, Riggs PD, Cheng X (1998) J Mol Biol 284:1491–1504

    Article  PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. Department of Chemistry and Biochemistry, The Center for Nanoscience, University of Missouri St. Louis, St. Louis, MO, 63121, USA

    Grigorios A. Papadakos & Cynthia M. Dupureur

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  1. Grigorios A. Papadakos
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  2. Cynthia M. Dupureur
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Correspondence to Cynthia M. Dupureur.

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Papadakos, G.A., Dupureur, C.M. Metal ion and DNA binding by single-chain PvuII endonuclease: lessons from the linker. J Biol Inorg Chem 16, 1269–1278 (2011). https://doi.org/10.1007/s00775-011-0814-7

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