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Molecular and Cellular Biology, May 1999, p. 3267-3277, Vol. 19, No. 5
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

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

B. K. Singleton,1 M. I. Torres-Arzayus,2 S. T. Rottinghaus,3 G. E. Taccioli,2 and P. A. Jeggo1,*

MRC Cell Mutation Unit, University of Sussex, Brighton BN1 9RR,1 and Wellcome CRC Institute and Department of Zoology, Cambridge University, Cambridge CB2 1QR,3 United Kingdom, and Department of Microbiology, School of Medicine, Boston University, Boston, Massachusetts 021182

Received 17 August 1998/Returned for modification 27 November 1998/Accepted 22 January 1999

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.

* Corresponding author. Mailing address: MRC Cell Mutation Unit, University of Sussex, Falmer, Brighton BN1 9RR, United Kingdom. Phone: 44 1273 678482. Fax: 44 1273 678121. E-mail: p.a.jeggo{at}sussex.ac.uk.

Molecular and Cellular Biology, May 1999, p. 3267-3277, Vol. 19, No. 5
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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