Skip to main content
SpringerLink
Log in

Characterization of a Single-Stranded DNA-Binding Protein from Pseudomonas aeruginosa PAO1

  • Published:
The Protein Journal Aims and scope Submit manuscript

Abstract

Single-stranded DNA-binding protein (SSB) plays an important role in DNA metabolism, such as in DNA replication, repair, and recombination, and is essential for cell survival. We characterized the single-stranded DNA (ssDNA)-binding properties of Pseudomonas aeruginosa PAO1 SSB (PaSSB) by using fluorescence quenching measurements and electrophoretic mobility shift analysis (EMSA). Analysis of purified PaSSB by gel filtration chromatography revealed a stable tetramer in solution. In fluorescence titrations, PaSSB bound 22–32 nucleotides (nt) per tetramer depending on salt concentration. Using EMSA, we characterized the stoichiometry of PaSSB complexed with a series of ssDNA homopolymers, and the size of the binding site was determined to be 29 ± 1 nt. Furthermore, EMSA results indicated that the dissociation constants of PaSSB for the first tetramer were less than those for the second tetramer. On the basis of these biophysical analyses, the ssDNA binding mode of PaSSB is expected to be noncooperative.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

Abbreviations

Ec :

Escherichia coli

Pa :

Pseudomonas aeruginosa PAO1

Mt :

Mycobacterium tuberculosis

Ms :

Mycobacterium smegmatis

Hp :

Helicobacter pylori

ssDNA:

Single-stranded DNA

SSB:

Single-stranded DNA-binding protein

SDS-PAGE:

Sodium dodecyl sulphate–polyacrylamide gel electrophoresis

EDTA:

Ethylenediamine tetraacetic acid

EMSA:

Electrophoretic mobility shift analysis

nt:

Nucleotides

K d :

The apparent dissociation constant

References

  1. Casas-Finet JR, Fischer KR, Karpel RL (1992) Proc Natl Acad Sci USA 89:1050–1054

    Article  CAS  Google Scholar 

  2. Chan KW, Lee YJ, Wang CH, Huang H, Sun YJ (2009) J Mol Biol 388:508–519

    Article  CAS  Google Scholar 

  3. Chase JW, Williams KR (1986) Annu Rev Biochem 55:103–136

    Article  CAS  Google Scholar 

  4. Curth U, Genschel J, Urbanke C, Greipel J (1996) Nucleic Acids Res 24:2706–2711

    Article  CAS  Google Scholar 

  5. Curth U, Greipel J, Urbanke C, Maass G (1993) Biochemistry 32:2585–2591

    Article  CAS  Google Scholar 

  6. Dabrowski S, Olszewski M, Piatek R, Brillowska-Dabrowska A, Konopa G, Kur J (2002) Microbiology 148:3307–3315

    CAS  Google Scholar 

  7. Fairall L, Buttinelli M, Panetta G (2000) In: Travers A, Buckle M (eds) DNA-protein interactions: a practical approach. Oxford University Press, New York, pp 65–74

  8. Fedorov R, Witte G, Urbanke C, Manstein DJ, Curth U (2006) Nucleic Acids Res 34:6708–6717

    Article  CAS  Google Scholar 

  9. Genschel J, Litz L, Thole H, Roemling U, Urbanke C (1996) Gene 182:137–143

    Article  Google Scholar 

  10. Haseltine CA, Kowalczykowski SC (2002) Mol Microbiol 43:1505–1515

    Article  CAS  Google Scholar 

  11. Huang CY, Chang YW, Chen WT (2008) Biochem Biophys Res Commun 375:220–224

    Article  CAS  Google Scholar 

  12. Huang CY, Hsu CH, Sun YJ, Wu HN, Hsiao CD (2006) Nucleic Acids Res 34:3878–3886

    Article  CAS  Google Scholar 

  13. Jedrzejczak R, Dauter M, Dauter Z, Olszewski M, Dlugolecka A, Kur J (2006) Acta Crystallogr D Biol Crystallogr 62:1407–1412

    Article  Google Scholar 

  14. Kelly TJ, Simancek P, Brush GS (1998) Proc Natl Acad Sci USA 95:14634–14639

    Article  CAS  Google Scholar 

  15. Kerr ID, Wadsworth RI, Cubeddu L, Blankenfeldt W, Naismith JH, White MF (2003) EMBO J 22:2561–2570

    Article  CAS  Google Scholar 

  16. Kim C, Snyder RO, Wold MS (1992) Mol Cell Biol 12:3050–3059

    CAS  Google Scholar 

  17. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG (2007) Bioinformatics 23:2947–2948

    Article  CAS  Google Scholar 

  18. Liu JH, Chang TW, Huang CY, Chen SU, Wu HN, Chang MC, Hsiao CD (2004) J Biol Chem 279:50465–50471

    Article  CAS  Google Scholar 

  19. Lohman TM, Ferrari ME (1994) Annu Rev Biochem 63:527–570

    CAS  Google Scholar 

  20. Madden TL, Tatusov RL, Zhang J (1996) Methods Enzymol 266:131–141

    Article  CAS  Google Scholar 

  21. Matos RG, Barbas A, Arraiano CM (2010) Protein J 29:394–397

    Article  CAS  Google Scholar 

  22. Meyer RR, Laine PS (1990) Microbiol Rev 54:342–380

    CAS  Google Scholar 

  23. Murzin AG (1993) EMBO J 12:861–867

    CAS  Google Scholar 

  24. Oakley GG, Patrick SM (2010) Front Biosci 15:883–900

    Article  CAS  Google Scholar 

  25. Olszewski M, Mickiewicz M, Kur J (2008) Arch Microbiol 190:79–87

    Article  CAS  Google Scholar 

  26. Raghunathan S, Kozlov AG, Lohman TM, Waksman G (2000) Nat Struct Biol 7:648–652

    Article  CAS  Google Scholar 

  27. Roy R, Kozlov AG, Lohman TM, Ha T (2007) J Mol Biol 369:1244–1257

    Article  CAS  Google Scholar 

  28. Roy R, Kozlov AG, Lohman TM, Ha T (2009) Nature 461:1092–1097

    Article  CAS  Google Scholar 

  29. Saikrishnan K, Jeyakanthan J, Venkatesh J, Acharya N, Sekar K, Varshney U, Vijayan M (2003) J Mol Biol 331:385–393

    Article  CAS  Google Scholar 

  30. Saikrishnan K, Manjunath GP, Singh P, Jeyakanthan J, Dauter Z, Sekar K, Muniyappa K, Vijayan M (2005) Acta Crystallogr D Biol Crystallogr 61:1140–1148

    Article  CAS  Google Scholar 

  31. Savvides SN, Raghunathan S, Futterer K, Kozlov AG, Lohman TM, Waksman G (2004) Protein Sci 13:1942–1947

    Article  CAS  Google Scholar 

  32. Schwarz G, Watanabe F (1983) J Mol Biol 163:467–484

    Article  CAS  Google Scholar 

  33. Shereda RD, Kozlov AG, Lohman TM, Cox MM, Keck JL (2008) Crit Rev Biochem Mol Biol 43:289–318

    Article  CAS  Google Scholar 

  34. Tsai KL, Huang CY, Chang CH, Sun YJ, Chuang WJ, Hsiao CD (2006) J Biol Chem 281:17400–17409

    Article  CAS  Google Scholar 

  35. Wadsworth RI, White MF (2001) Nucleic Acids Res 29:914–920

    Article  CAS  Google Scholar 

  36. Wang CC, Tsau HW, Chen WT, Huang CY (2010) Protein J 29:445–452

    Article  CAS  Google Scholar 

  37. Winsor GL, Lo R, Sui SJ, Ung KS, Huang S, Cheng D, Ching WK, Hancock RE, Brinkman FS (2005) Nucleic Acids Res 33:D338–D343

    Article  CAS  Google Scholar 

  38. Witte G, Fedorov R, Curth U (2008) Biophys J 94:2269–2279

    Article  CAS  Google Scholar 

  39. Witte G, Urbanke C, Curth U (2003) Nucleic Acids Res 31:4434–4440

    Article  CAS  Google Scholar 

  40. Witte G, Urbanke C, Curth U (2005) Nucleic Acids Res 33:1662–1670

    Article  CAS  Google Scholar 

  41. Wold MS (1997) Annu Rev Biochem 66:61–92

    Article  CAS  Google Scholar 

  42. Yang C, Curth U, Urbanke C, Kang C (1997) Nat Struct Biol 4:153–157

    Article  CAS  Google Scholar 

  43. Zhao WH, Hu ZQ (2010) Crit Rev Microbiol 36:245–258

    Article  CAS  Google Scholar 

Download references

Acknowledgments

We thank Ms. Hui-Chuan Hsieh for constructing the pET21b-PaSSB. This research was supported a grant from the National Research Program for Genome Medicine (NSC 99-3112-B-040-001 to C.Y. Huang).

Author information

Authors and Affiliations

  1. Department of Biomedical Sciences, Chung Shan Medical University, No. 110, Sec.1, Chien-Kuo N. Rd., Taichung City, Taiwan

    Hau-Chern Jan, Yen-Ling Lee & Cheng-Yang Huang

  2. Department of Medical Research, Chung Shan Medical University Hospital, No. 110, Sec.1, Chien-Kuo N. Rd., Taichung City, Taiwan

    Cheng-Yang Huang

Authors
  1. Hau-Chern Jan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yen-Ling Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Cheng-Yang Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Cheng-Yang Huang.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Jan, HC., Lee, YL. & Huang, CY. Characterization of a Single-Stranded DNA-Binding Protein from Pseudomonas aeruginosa PAO1. Protein J 30, 20–26 (2011). https://doi.org/10.1007/s10930-010-9297-6

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10930-010-9297-6

Keywords

Search

Navigation