Nucleic Acids Research, 2003, Vol. 31, No. 20 5848-5857
© 2003 Oxford University Press
Modification of the ionizing radiation response in living cells by an scFv against the DNA-dependent protein kinase
Institute of Molecular Medicine and Genetics and 1 Department of Radiology, Medical College of Georgia, Augusta, GA 30912, USA
*To whom correspondence should be addressed. Tel: +1 706 721 8756; Fax: +1 706 721 8752; Email: wdynan{at}mail.mcg.edu
The non-homologous end joining pathway uses pre-existing proteins to repair DNA double-strand breaks induced by ionizing radiation. Here we describe manipulation of this pathway in living cells using a newly developed tool. We generated a single chain antibody variable fragment (scFv) that binds to the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a key enzyme in the pathway. In contrast to existing pharmacologic inhibitors, the scFv binds a newly defined regulatory site outside the kinase catalytic domain. Although the scFv inhibits kinase activity only modestly, it completely blocks DNA end joining in a cell-free system. Microinjection of the scFv sensitizes human cells to radiation, as measured by a reduction in efficiency of colony formation and induction of apoptosis at an otherwise sublethal dose of 1.5 Gy. The scFv blocks non-homologous end joining in situ at a step subsequent to histone 
-H2AX focus formation but preceding -H2AX dephosphorylation. Blockage occurs in cells exposed to as little as 0.1 Gy, indicating that DNA-PKcs is essential for double-strand break repair even at low radiation doses. The ability to modify the radiation response in situ in living cells provides a link between biochemical, genetic and cytologic approaches to the study of double-strand break repair intermediates.
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