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Defining the minimal domain of Ku80 for interaction with Ku70
O Osipovich1, S K Durum, K Muegge
1Laboratory of Molecular Immunoregulation, NCI, National Institutes of Health, Frederick, Maryland 21702-1201, USA.
Insights
The Ku protein
Area of Science:
- Molecular Biology
- DNA Repair Mechanisms
- Protein Interactions
Background:
- The Ku protein complex is essential for DNA double-strand break repair and V(D)J recombination.
- Ku is a heterodimer composed of 80-kDa (Ku80) and 70-kDa (Ku70) subunits.
- Understanding Ku subunit interactions is key to elucidating its function in DNA repair.
Purpose of the Study:
- To analyze the interaction domains of Ku80 and Ku70 subunits.
- To identify potential novel binding partners for the Ku heterodimer and individual subunits.
- To define the minimal functional interaction region of Ku80 with Ku70.
Main Methods:
- Yeast two-hybrid system screening of a human cDNA library.
- Analysis of subunit interactions using individual and heterodimer screening.
- In vitro co-translation followed by immunoprecipitation assays.
Main Results:
- Ku80 and Ku70 exclusively form heterodimers, with no evidence of homodimerization.
- Ku80 interacts with only one Ku70 molecule at a time.
- A specific 28-amino acid region (residues 449-477) in Ku80 was identified as the minimal interaction domain for Ku70, crucial for binding.
Conclusions:
- The Ku heterodimer is formed by specific interactions between Ku80 and Ku70 subunits.
- A novel, previously unrecognized interaction domain in Ku80 mediates binding to Ku70.
- This defined interaction domain is critical for Ku heterodimer formation and likely its function in DNA repair.
Abstract:
The Ku protein has a critical function in the repair of double-strand DNA breaks induced for example by ionizing radiation or during VDJ recombination. Ku serves as the DNA-binding subunit of the DNA-dependent kinase and is a heterodimeric protein composed of 80- and 70-kDa subunits. We used the two-hybrid system to analyze the interaction domains of the Ku subunits and to identify possible additional partners for Ku. Screening a human cDNA library with the Ku heterodimer did not reveal any novel partners. Screening with the individual subunits, we detected only Ku70 clones interacting with Ku80 and only Ku80 clones interacting with Ku70, indicating that these are the primary partners for one another. Ku80 and Ku70 formed only heterodimers and did not homodimerize. Ku80 was restricted to interacting with just one Ku70 molecule at a time. The minimal functional interaction domain of Ku80 that interacted with Ku70 was defined. It consisted of a 28-amino acid region extending from amino acid 449 to 477. This region was crucial for interaction with Ku70, since mutation within this critical site at amino acids 453 and 454 abrogated the ability to interact with Ku70. We furthermore verified that the same region is crucial for interaction with Ku70 using in vitro co-translation of both subunits followed by an immunoprecipitation with anti-Ku70 antibodies. This interaction domain of Ku80 does not contain any motif previously recognized in protein-protein interactions.