The C Terminus of Ku80 Activates the DNA-Dependent Protein Kinase Catalytic Subunit

Abstract

Ku is a heterodimeric protein with double-stranded DNA end-binding activity that operates in the process of nonhomologous end joining. Ku is thought to target the DNA-dependent protein kinase (DNA-PK) complex to the DNA and, when DNA bound, can interact and activate the DNA-PK catalytic subunit (DNA-PKcs). We have carried out a 3′ deletion analysis of Ku80, the larger subunit of Ku, and shown that the C-terminal 178 amino acid residues are dispensable for DNA end-binding activity but are required for efficient interaction of Ku with DNA-PKcs. Cells expressing Ku80 proteins that lack the terminal 178 residues have low DNA-PK activity, are radiation sensitive, and can recombine the signal junctions but not the coding junctions during V(D)J recombination. These cells have therefore acquired the phenotype of mouse SCID cells despite expressing DNA-PKcs protein, suggesting that an interaction between DNA-PKcs and Ku, involving the C-terminal region of Ku80, is required for DNA double-strand break rejoining and coding but not signal joint formation. To gain further insight into important domains in Ku80, we report a point mutational change in Ku80 in the defective xrs-2 cell line. This residue is conserved among species and lies outside of the previously reported Ku70-Ku80 interaction domain. The mutational change nonetheless abrogates the Ku70-Ku80 interaction and DNA end-binding activity.

ACKNOWLEDGMENTS

We thank S. P. Jackson and W. H. Reeves for providing antibodies. We thank S. P. Jackson, D. Gell, H. Beamish, and A. R. Lehman for advice and critical reading of the manuscript.

This work was supported by a grant from the Kay Kendall Leukaemia Fund. Additional work in the P.A.J. laboratory is funded by European Union grant F13PCT920007, by the Human Frontiers Science Programme, and by a grant from the UKCCCR Radiation Research Programme. G.E.T. is a special fellow of the Leukemia Society of America and is supported by grant CA76409 from the National Cancer Institute. S.T.R. was supported by a Marshall Scholarship.