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DOE Lowdose Radiation Program Workshop IV

Abstract

Title: Repair of sparse double-strand breaks induced at low radiation doses: modification of the radiation response in living cells using intracellular single-chain antibodies

Authors: William S. Dynan*, Shuyi Li*, Yoshihiko Takeda*, Stéphanie Wragg*, Andrew Phillips*, and John Barrett+

Institutions: *Institute of Molecular Medicine and Genetics and +Department of Radiology Medical College of Georgia, Augusta, Georgia *Presenting author
E-mail:wdynan@mail.mcg.edu. Telephone: 706-721-8756; Fax: 706-721-8752
Mailing address: IMMAG CB-2803, Medical College of Georgia, Augusta, GA 30912.

The genotoxic effects of ionizing radiation are attributable, in large part, to DNA double-strand breaks (DSBs). Efficient cellular DSB repair pathways serve to protect against these effects. There are at least two major DSB repair pathways, one based on homologous recombination and the other on direct, nonhomologous end joining. The mechanisms and relative contributions of each pathway are best understood at high radiation doses. Until recently, tools for investigating pathways used for repair of the sparse DSBs that are induced at low radiation doses have been lacking.

Here we describe the isolation and characterization of a single-chain antibody variable fragment (scFv) that binds to the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a key enzyme in the nonhomologous end-joining pathway of repair. The scFv is highly specific for DNA-PKcs versus related cellular kinases. It recognizes a 25-residue linear epitope, located in sequences unique to DNA-PKcs, well outside the conserved kinase catalytic domain. The scFv inhibits kinase activity only modestly, but completely blocks DNA-PKcs-dependent nonhomologous end joining in a cell-free assay system. Microinjection of the scFv sensitizes human cells to radiation-induced cell death, as measured by a reduction in efficiency of colony
formation and induction of apoptosis at an otherwise sublethal radiation dose.

The effect of the antibody on repair of sparse DSBs, induced at low doses, was evaluated by measuring the kinetics of induction and decay of histone y-H2AX foci. Such foci, which can be observed by immunostaining of cell nuclei, have previously been shown to correlate precisely with unrepaired DSBs and thus serve as a surrogate marker for DSBs induced at low radiation doses. We show that the scFv does not affect induction of y-H2AX foci at a 0.1 Gy dose of low LET radiation, but does prevent or delay their disappearance. These results suggest that the scFv
blocks the nonhomologous end joining pathway at a step subsequent to histone y-H2AX focus formation but preceding y-H2AX focus dephosphorylation. The results provide the first direct evidence that DNA-PKcs is involved in repair of the sparse DSBs induced at low radiation doses. They complement and extend previous findings, based on analysis of a human cell mutant, that DNA ligase IV, another nonhomologous end-joining enzyme, is also required for repair of damage induced at low doses.

The ability to modify the radiation response in situ in living cells using intracellular antibodies provides a link between biochemical, genetic, and cytologic approaches to the study of DSB repair. The approach is potentially general, and is independent of the ability to obtain mutants affecting a particular pathway. It is thus applicable to study the role of DNA-PKcs in a variety of other human cell types and in organisms that are not amenable to genetic manipulation. By using scFvs directed against other candidate repair proteins, the approach can also be extended to investigate the role of these proteins in the-low dose response.

SK-MEL-28 cells were injected with scFv18-2(directed against DNA-PKcs) or with sdFv 147 (a control antibody.