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Checkpoint activation in response to double-strand breaks requires the Mre11/Rad50/Xrs2 complex
M Grenon1, C Gilbert, N F Lowndes
1ICRF Clare Hall Laboratories, CDC Laboratory, Blanche Lane, South Mimms, Potters Bar, Hertfordshire EN6 3LD, UK.
Abstract:
Studies of human Nijmegen breakage syndrome (NBS) cells have led to the proposal that the Mre11/Rad50/ NBS1 complex, which is involved in the repair of DNA double-strand breaks (DSBs), might also function in activating the DNA damage checkpoint pathways after DSBs occur. We have studied the role of the homologous budding yeast complex, Mre11/Rad50/Xrs2, in checkpoint activation in response to DSB-inducing agents. Here we show that this complex is required for phosphorylation and activation of the Rad53 and Chk1 checkpoint kinases specifically in response to DSBs. Consistent with defective Rad53 activation, we observed defective cell-cycle delays after induction of DSBs in the absence of Mre11. Furthermore, after gamma-irradiation phosphorylation of Rad9, which is an early event in checkpoint activation, is also dependent on Mre11. All three components of the Mre11/Rad50/Xrs2 complex are required for activation of Rad53, however, the Ku80, Rad51 or Rad52 proteins, which are also involved in DSB repair, are not. Thus, the integrity of the Mre11/Rad50/Xrs2 complex is specifically required for checkpoint activation after the formation of DSBs.
Insights
The Mre11/Rad50/Xrs2 complex is crucial for activating DNA damage checkpoints after double-strand breaks (DSBs). This complex is specifically required for checkpoint activation, unlike other DSB repair proteins.
Area of Science:
- Molecular Biology
- Cellular Biology
- Genetics
Background:
- The Mre11/Rad50/NBS1 complex in humans is implicated in DNA double-strand break (DSB) repair and DNA damage checkpoint activation.
- The homologous budding yeast complex, Mre11/Rad50/Xrs2, has been investigated for its role in checkpoint activation following DSBs.
Purpose of the Study:
- To investigate the role of the Mre11/Rad50/Xrs2 complex in activating DNA damage checkpoint pathways in response to DSBs in yeast.
- To determine if the Mre11/Rad50/Xrs2 complex is specifically required for checkpoint activation after DSB formation.
Main Methods:
- Studied the phosphorylation and activation of checkpoint kinases Rad53 and Chk1 in response to DSB-inducing agents.
- Assessed cell-cycle delays after inducing DSBs in yeast cells lacking Mre11.
- Examined the phosphorylation of Rad9 after gamma-irradiation in the presence and absence of Mre11.
- Investigated the requirement of Mre11/Rad50/Xrs2 complex components versus other DSB repair proteins (Ku80, Rad51, Rad52) for checkpoint activation.
Main Results:
- The Mre11/Rad50/Xrs2 complex is essential for the phosphorylation and activation of Rad53 and Chk1 checkpoint kinases specifically in response to DSBs.
- Absence of Mre11 leads to defective Rad53 activation and impaired cell-cycle delays following DSB induction.
- Phosphorylation of Rad9, an early checkpoint activation event, is dependent on Mre11 after gamma-irradiation.
- All three components of the Mre11/Rad50/Xrs2 complex are necessary for Rad53 activation, but Ku80, Rad51, or Rad52 are not.
Conclusions:
- The integrity of the Mre11/Rad50/Xrs2 complex is specifically required for the activation of DNA damage checkpoints after the formation of DSBs.
- This finding highlights a specific role for the Mre11/Rad50/Xrs2 complex in sensing and signaling DSBs to initiate cell-cycle arrest.