C-Abl downregulates the slow phase of double-strand break repair

V Meltser,Y Shaul,M Ben-Yehoyada,N Reuven

doi:10.1038/cddis.2009.21

V Meltser, Y Shaul + Show 2 more

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https://doi.org/10.1038/cddis.2009.21

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Abstract

c-Abl tyrosine kinase is activated by agents that induce double-strand DNA breaks (DSBs) and interacts with key components of the DNA damage response and of the DSB repair machinery. However, the functional significance of c-Abl in these processes, remained unclear. In this study, we demonstrate, using comet assay and pulsed-field gel electrophoresis, that c-Abl inhibited the repair of DSBs induced by ionizing radiation, particularly during the second and slow phase of DSB repair. Pharmacological inhibition of c-Abl and c-Abl depletion by siRNA-mediated knockdown resulted in higher DSB rejoining. c-Abl null MEFs exhibited higher DSB rejoining compared with cells reconstituted for c-Abl expression. Abrogation of c-Abl kinase activation resulted in higher H2AX phosphorylation levels and higher numbers of post-irradiation γH2AX foci, consistent with a role of c-Abl in DSB repair regulation. In conjunction with these findings, transient abrogation of c-Abl activity resulted in increased cellular radioresistance. Our findings suggest a novel function for c-Abl in inhibition of the slow phase of DSB repair.

Highlights

Activation of c-Abl by DNA damage and abrogation of this activation by STI-571 pretreatment was verified by assessing the level of c-Abl tyrosine autophosphorylation (Figure 1a), a modification known to be important for c-Abl kinase activation.[20,21]
To assess the effect of c-Abl kinase activity on DSB repair following g-irradiation, the comet assay,[22,23] which provides an estimate for the degree of DSB rejoining in individual cells, was used. wt MEFs were incubated in the presence or absence of STI-571, g-irradiated, harvested following 24 h of incubation and subjected to the comet assay (Figure 1b)
To establish the c-Abl specificity of this effect, we used c-Abl null mouse embryonic fibroblasts reconstituted for c-Abl expression.[9] c-Abl expression was verified by WB (Figure 1c, right panel)

Summary

Introduction

To assess the effect of c-Abl kinase activity on DSB repair following g-irradiation, the comet assay,[22,23] which provides an estimate for the degree of DSB rejoining in individual cells, was used.

Results

Conclusion