Modification of the ionizing radiation response in living cells by an scFv against the DNA-dependent protein kinase.
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 gamma-H2AX focus formation but preceding gamma-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.
Li
Shuyi
S
Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA 30912, USA.
Takeda
Yoshihiko
Y
Wragg
Stéphanie
S
Barrett
John
J
Phillips
Andrew
A
Dynan
William S
WS
D017209:Apoptosis / Q000187:drug effects* / Q000528:radiation effects; D001666:Binding Sites, Antibody; D020134:Catalytic Domain; D002455:Cell Division / Q000187:drug effects / Q000528:radiation effects; D002461:Cell Line, Transformed; D045744:Cell Line, Tumor; D002474:Cell-Free System / Q000187:drug effects / Q000378:metabolism / Q000528:radiation effects; D004247:DNA / Q000187:drug effects / Q000235:genetics / Q000528:radiation effects; D004249:DNA Damage; D004260:DNA Repair; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins; D006367:HeLa Cells; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D009687:Nuclear Proteins; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000276:immunology* / Q000378:metabolism; D011839:Radiation, Ionizing; D011995:Recombination, Genetic / Q000187:drug effects / Q000528:radiation effects
D004268:DNA-Binding Proteins; D007128:Immunoglobulin Fragments; D009687:Nuclear Proteins; C055966:immunoglobulin Fv; D004247:DNA; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases