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Related Experiment Videos

Sequence-specific binding of Ku autoantigen to single-stranded DNA

H Torrance1, W Giffin, D J Rodda

  • 1Graduate Program in Biochemistry, University of Ottawa, Loeb Institute for Medical Research, Ottawa Civic Hospital, Ottawa, Ontario K1Y 4E9, Canada.

The Journal of Biological Chemistry
|August 8, 1998
PubMed
Summary

Ku antigen binds to single-stranded DNA regulatory elements, influencing gene transcription. This protein kinase plays a key role in regulating viral gene expression, with specific subunits showing distinct binding behaviors.

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Area of Science:

  • Molecular Biology
  • Virology
  • Biochemistry

Background:

  • Glucocorticoid-induced transcription of mouse mammary tumor virus (MMTV) is repressed by Ku antigen/DNA-dependent protein kinase (DNA-PK).
  • This repression occurs via a specific DNA sequence element (NRE1) within the viral long terminal repeat.
  • Nuclear factors binding to single-stranded NRE1 may also regulate transcription.

Purpose of the Study:

  • To identify and characterize nuclear factors that bind to the single-stranded NRE1 element.
  • To investigate the role of Ku antigen in binding to single-stranded DNA regulatory elements.
  • To elucidate the subunit-specific interactions of Ku antigen with single-stranded DNA.

Main Methods:

  • Separation of nuclear extracts from Jurkat T cells to isolate DNA-binding activities.

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  • Biochemical assays to characterize Ku antigen binding to single-stranded and double-stranded DNA.
  • Cross-linking studies to identify Ku subunits interacting with NRE1.
  • Analysis of Ku antigen's binding kinetics and affinity.

    • Main Results:

    • Ku antigen was identified as a major component of the upper-stranded NRE1 binding activity.
    • Ku antigen exhibits sequence-specific binding to single-stranded DNA, with preferences similar to double-stranded DNA binding.
    • Ku70 cross-linked to both single- and double-stranded NRE1, while Ku80 preferentially cross-linked to single-stranded NRE1.
    • Ku80 cross-linking to double-stranded DNA was induced by Mg2+ and ATP, without complete strand unwinding.

    Conclusions:

    • Ku antigen binds to single-stranded DNA regulatory elements like NRE1.
    • The differential subunit cross-linking suggests distinct roles for Ku70 and Ku80 in single-stranded DNA interactions.
    • Ku antigen's interaction with single-stranded DNA may be crucial for its role in transcriptional repression of MMTV.