Abstract
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 γ-H2AX focus formation but preceding γ-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 doublestrand break repair intermediates.
Original language | English (US) |
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Pages (from-to) | 5848-5857 |
Number of pages | 10 |
Journal | Nucleic Acids Research |
Volume | 31 |
Issue number | 20 |
DOIs | |
State | Published - Oct 15 2003 |
ASJC Scopus subject areas
- Genetics