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Related Experiment Videos

Complex formation between phage phi 29 single-stranded DNA binding protein and DNA

M S Soengas1, J A Esteban, M Salas

  • 1Centro de Biología Molecular Severo Ochoa (CSIC-UAM) Universidad Autónoma, Cantoblanco, Madrid, Spain.

Journal of Molecular Biology
|June 3, 1994
PubMed
Summary

Bacteriophage phi 29 single-stranded DNA binding protein (SSB) forms homogeneous complexes with DNA, showing high binding affinity and a specific nucleotide-to-protein ratio. These binding parameters are crucial for viral DNA replication.

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Area of Science:

  • Molecular Biology
  • Virology
  • Biophysics

Background:

  • Bacteriophage phi 29 gene 5 encodes a single-stranded DNA (ssDNA) binding protein (SSB).
  • This SSB is essential for stimulating viral DNA replication.

Purpose of the Study:

  • To structurally characterize the complex between phi 29 SSB and ssDNA.
  • To elucidate the binding parameters and functional significance of phi 29 SSB in viral DNA replication.

Main Methods:

  • Electron microscopy
  • Band-shift assays
  • Nuclease digestion
  • Intrinsic fluorescence monitoring

Main Results:

  • Phi 29 SSB binds ssDNA forming homogeneous, continuous arrays.

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  • ssDNA binding causes significant fluorescence quenching (Qmax = 94.6%).
  • Stoichiometry is 3.4 nucleotides per SSB monomer, independent of salt concentration.
  • Binding affinity (Keff) is high (2.2 x 10^5 M-1) at low salt, decreasing slightly with increased salt.

    • Conclusions:

    • Phi 29 SSB exhibits a single, high-affinity binding mode to ssDNA.
    • The characterized binding parameters likely play a key role in bacteriophage phi 29 DNA replication.