Abstract
Double strand break repair and V(D)J recombination in mammalian cells require the function of the Ku protein complex and the DNA-dependent protein kinase. The DNA-dependent protein kinase is targeted to DNA through its interaction with the Ku protein complex, and thus the specificity of template recognition in the repair and recombination reactions depend on Ku. We have studied Ku binding to DNA using competitive gel shift analysis. We find that Ku bound to one DNA molecule can transfer directly to another DNA molecule when the two DNA molecules have homologous ends containing a minimum of four matched bases. This remarkable reaction can give a false impression of sequence specificity of Ku DNA binding under certain assay conditions. A model is proposed for the DNA binding function of Ku on the basis of these results and the discovery of a novel type of DNA-Ku complex formed at high Ku/DNA ratios is discussed.
Highlights
The Ku autoantigen is a nuclear DNA-binding protein composed of two subunits, one of approximately 70 kDa and the other 80 kDa (Ku70 and Ku80, respectively) (1, 2)
These results suggest that Ku and DNA-PK are essential for DNA double strand break repair and V(D)J recombination
Ku is a DNA-binding protein known to interact with the ends of double-stranded DNA fragments, but there is some debate in the literature about whether Ku can bind DNA in a sequencedependent manner
Summary
Reagents and Proteins—Bacterially expressed, wild type human p53 was purified as described previously (34). The protein was applied to a Q50 anion exchange column (150 ml) equilibrated in buffer B containing 10 mM KCl and eluted with a linear gradient of 10 –700 mM KCl in buffer B. 20 l of DNA binding buffer (40 mM HEPES, pH 7.6, containing 20% (v/v) glycerol, 5 mM dithiothreitol, 1 mg/ml bovine serum albumin, 0.1% Triton X-100, 50 mM KCl, 0.5 mM EDTA, 10 mM MgCl2) was mixed with the relevant radiolabeled DNA (ϳ2– 4 ng) and the indicated amounts of purified Ku protein. Reaction products were loaded onto a 4% polyacrylamide gel containing 0.56 ϫ Tris borate/EDTA (TBE) and 0.1% Triton X-100, which had undergone pre-electrophoresis at 150 V for 5 min at 4 °C in a 0.33 ϫ TBE/0.1% Triton X-100 running buffer. Gels were dried and exposed to x-ray film at Ϫ70 °C
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