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

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.
• 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.