Published online 18 May 2004
Nucleic Acids Research, 2004, Vol. 32, No. 9 2776-2784
© 2004 Oxford University Press
Visualization of inositol phosphate-dependent mobility of Ku: depletion of the DNA–PK cofactor InsP6 inhibits Ku mobility
Molecular Immunogenetics Program, Oklahoma Medical Research Foundation, 825 NE 13th Street, MS 17, Oklahoma City, OK 73104, USA and 1 School of Life Sciences, The University of Dundee, Dundee, Scotland, UK
*To whom correspondence should be addressed. Tel: +1 405 271 7393; Fax: +1 405 271 8237; Email: william-rodgers{at}omrf.ouhsc.edu
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
Received as resubmission March 14, 2004; Revised and Accepted April 15, 2004
Repair of DNA double-strand breaks (DSBs) in mammalian cells by nonhomologous end-joining (NHEJ) is initiated by the DNA–PK protein complex. Recent studies have shown inositol hexakisphosphate (InsP6) is a potent cofactor for DNA–PK activity in NHEJ. Specifically, InsP6 binds to the Ku component of DNA–PK, where it induces a conformational change and a corresponding increase in DNA end-joining activity. However, the effect of InsP6 on the dynamics of Ku, such as its mobility in the nucleus, is unknown. Importantly, these dynamics reflect the character of Ku’s interactions with other molecules. To address this question, the diffusion of Ku was measured by fluorescence photobleaching experiments using cells expressing green fluorescent protein (GFP)-labeled Ku. InsP6 was depleted by treating cells with calmodulin inhibitors, which included the compounds W7 and chlorpromazine. These treatments caused a 50% reduction in the mobile fraction of Ku–GFP, and this could be reversed by replenishing cells with InsP6. By expressing deletion mutants of Ku–GFP, it was determined that its W7-sensitive region occurred at the N-terminus of the dimerization domain of Ku70. These results therefore show that InsP6 enhances Ku mobility through a discrete region of Ku70, and modulation of InsP6 levels in cells represents a potential avenue for regulating NHEJ by affecting the dynamics of Ku and hence its interaction with other nuclear proteins.
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