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Slx4 and Rtt107 control checkpoint signalling and DNA resection at double-strand breaks
Diego Dibitetto1, Matteo Ferrari1, Chetan C Rawal1
1Department of Biosciences, University of Milan, 20133, Milano, Italy.
Abstract:
The DNA damage checkpoint pathway is activated in response to DNA lesions and replication stress to preserve genome integrity. However, hyper-activation of this surveillance system is detrimental to the cell, because it might prevent cell cycle re-start after repair, which may also lead to senescence. Here we show that the scaffold proteins Slx4 and Rtt107 limit checkpoint signalling at a persistent double-strand DNA break (DSB) and at uncapped telomeres. We found that Slx4 is recruited within a few kilobases of an irreparable DSB, through the interaction with Rtt107 and the multi-BRCT domain scaffold Dpb11. In the absence of Slx4 or Rtt107, Rad9 binding near the irreparable DSB is increased, leading to robust checkpoint signalling and slower nucleolytic degradation of the 5' strand. Importantly, in slx4Δ sae2Δ double mutant cells these phenotypes are exacerbated, causing a severe Rad9-dependent defect in DSB repair. Our study sheds new light on the molecular mechanism that coordinates the processing and repair of DSBs with DNA damage checkpoint signalling, preserving genome integrity.
Insights
Scaffold proteins Slx4 and Rtt107 limit DNA damage checkpoint signaling. Their absence causes increased Rad9 binding and defects in double-strand break repair, preserving genome integrity.
Area of Science:
- Molecular Biology
- Cell Biology
- Genetics
Background:
- The DNA damage checkpoint pathway maintains genome integrity by responding to DNA lesions.
- Hyper-activation of this pathway can hinder cell cycle restart and lead to senescence.
- Understanding checkpoint regulation is crucial for preventing genomic instability.
Purpose of the Study:
- To investigate the role of scaffold proteins Slx4 and Rtt107 in regulating DNA damage checkpoint signaling.
- To elucidate the molecular mechanisms by which Slx4 and Rtt107 limit checkpoint activation at persistent double-strand DNA breaks (DSBs).
Main Methods:
- Recruitment assays of Slx4 to irreparable DSBs.
- Analysis of Rad9 binding and DNA repair in wild-type and mutant cells (slx4Δ, rtt107Δ, slx4Δ sae2Δ).
- Assessment of nucleolytic degradation of the 5' strand at DSBs.
Main Results:
- Slx4 and Rtt107 limit checkpoint signaling at persistent DSBs and uncapped telomeres.
- Slx4 is recruited near irreparable DSBs via interactions with Rtt107 and Dpb11.
- Absence of Slx4 or Rtt107 increases Rad9 binding, enhances checkpoint signaling, and slows 5' strand degradation.
- Double mutants (slx4Δ sae2Δ) show exacerbated phenotypes and severe Rad9-dependent DSB repair defects.
Conclusions:
- Slx4 and Rtt107 act as negative regulators of the DNA damage checkpoint.
- These proteins coordinate DSB processing and repair with checkpoint signaling to maintain genome stability.
- Dysregulation of this pathway can lead to significant defects in DNA repair.
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