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) |
|---|---|
| Pages (from-to) | 5848-5857 |
| Number of pages | 10 |
| Journal | Nucleic Acids Research |
| Volume | 31 |
| Issue number | 20 |
| DOIs | |
| State | Published - Oct 15 2003 |
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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 journal › Article
}
TY - JOUR
T1 - Modification of the ionizing radiation response in living cells by an scFv against the DNA-dependent protein kinase
AU - Li, Shuyi
AU - Takeda, Yoshihiko
AU - Wragg, Stéphanie
AU - Barrett, John Thomas
AU - Phillips, Andrew
AU - Dynan, William S.
PY - 2003/10/15
Y1 - 2003/10/15
N2 - 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.
AB - 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.
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UR - http://www.scopus.com/inward/citedby.url?scp=0344442425&partnerID=8YFLogxK
U2 - 10.1093/nar/gkg775
DO - 10.1093/nar/gkg775
M3 - Article
C2 - 14530433
AN - SCOPUS:0344442425
VL - 31
SP - 5848
EP - 5857
JO - Nucleic Acids Research
JF - Nucleic Acids Research
SN - 0305-1048
IS - 20
ER -