A21
Radiation-induced recruitment and phosphorylation of cohesin in human cells
Christina Bauerschmidt1, David L Stevens1, Mark A Hill1, Kai Rothkamm2, Thomas Helleday1
1CRUK-MRC Gray Institute for Radiation Oncology & Biology, Oxford, UK, 2Health Protection Agency, Centre for Radiation, Chemical & Environmental Hazards, Chilton, UK
Background
The cohesion between sister chromatids depends on cohesin, a heterohexameric protein complex. SMC1 and SMC3 form heterodimers and are joined together by Rad21 which also binds to either SA1 or SA2.
Cohesin was previously reported to accumulate at sites of enzymatically-induced DNA double-strand breaks in yeast and in mammalian cells at sub-nuclear regions damaged by high energy lasers.
We wanted to test whether cohesin is recruited to and phosphorylated at sites of IR-induced DNA damage in human cells.
Method
HeLa cells were exposed to conventional X-rays or partially shielded ultra-soft X-rays. Immunofluorescence, Western blotting and immunoprecipitation were used to determine relocalisation and phosphorylation of cohesin subunits following irradiation. RNAi and mutant cell lines were used to identify factors mediating this response.
Results
Recruitment of the cohesin factor Rad21 to sites of X-ray-induced DNA damage was observed in G2-phase cells, but not in G1, and only when DNA damage was concentrated in 1 μm-wide stripes across the nucleus, generated by partially shielded ultra-soft X-rays.
SMC1 and SMC3 were phosphorylated following IR. These phosphorylations occurred at cohesin complexes located at the damaged site throughout the cell cycle and depended on ATM.
Individual RNAi knockdown of H2AX, 53BP1 and MDC1 had no effect on the formation of SMC1pS966 whereas levels of SMC3pS1083 were reduced to about 50%. Both phosphorylation events were severely reduced when all three damage response mediators were targeted simultaneously.
Conclusion
Human cohesin is involved in the cellular response to IR-induced DNA damage and promotes DNA double-strand break repair.
