Modification of the ionizing radiation response in living cells by an scFv against the DNA-dependent protein kinase

Shuyi Li, Yoshihiko Takeda, Stéphanie Wragg, John Thomas Barrett, Andrew Phillips, William S. Dynan

Research output: Contribution to journalArticle

23 Citations (Scopus)

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 languageEnglish (US)
Pages (from-to)5848-5857
Number of pages10
JournalNucleic Acids Research
Volume31
Issue number20
DOIs
StatePublished - Oct 15 2003

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DNA-Activated Protein Kinase
Ionizing Radiation
Catalytic Domain
Radiation
Phosphotransferases
Single-Chain Antibodies
Double-Stranded DNA Breaks
Cell-Free System
Microinjections
Histones
Molecular Biology
Apoptosis
DNA
Enzymes
Proteins

ASJC Scopus subject areas

  • Genetics

Cite this

Modification of the ionizing radiation response in living cells by an scFv against the DNA-dependent protein kinase. / Li, Shuyi; Takeda, Yoshihiko; Wragg, Stéphanie; Barrett, John Thomas; Phillips, Andrew; Dynan, William S.

In: Nucleic Acids Research, Vol. 31, No. 20, 15.10.2003, p. 5848-5857.

Research output: Contribution to journalArticle

Li, Shuyi ; Takeda, Yoshihiko ; Wragg, Stéphanie ; Barrett, John Thomas ; Phillips, Andrew ; Dynan, William S. / Modification of the ionizing radiation response in living cells by an scFv against the DNA-dependent protein kinase. In: Nucleic Acids Research. 2003 ; Vol. 31, No. 20. pp. 5848-5857.
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