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Sae2 controls Mre11 endo- and exonuclease activities by different mechanisms

Tomoki Tamai1, Giordano Reginato2, Ryusei Ojiri1

  • 1Faculty of Advanced Bioscience, Graduate School of Agriculture, Kindai University, Nara City, Nara, 631-8505, Japan.

Nature Communications
|August 22, 2024
PubMed

Insights

Sae2 protein controls DNA repair by stimulating the Mre11-Rad50-Xrs2 (MRX) complex. This study reveals Sae2 uses distinct mechanisms to activate MRX

Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • DNA double-strand breaks (DSBs) are critical DNA lesions that threaten cell survival and genomic integrity.
  • The Mre11-Rad50-Xrs2 (MRX) complex, along with Sae2, plays a crucial role in initiating DSB repair in yeast.
  • Sae2 is known to stimulate both endonuclease and 3'-5' exonuclease activities of the MRX complex, but the regulatory mechanisms are not fully understood.

Purpose of the Study:

  • To elucidate the mechanisms by which Sae2 controls the distinct nuclease activities of the MRX complex.
  • To investigate the roles of MRX-Sae2 endonuclease and exonuclease activities in specific DSB repair processes, such as Spo11 removal and hairpin resolution.
  • To identify specific mutations that can differentiate the functions of MRX-Sae2 nuclease activities.

Main Methods:

  • A combined genetic and biochemical approach was employed.
  • A novel separation-of-function mutation, rad50-C47, was identified and characterized.
  • Enzyme activity assays were performed to assess MRX endonuclease and exonuclease functions in vitro and in vivo.

Main Results:

  • The rad50-C47 mutation specifically impairs Sae2-dependent MRX 3'-5' exonuclease activity, while preserving endonuclease activity.
  • Both MRX endonuclease and 3'-5' exonuclease activities are essential for releasing Spo11 from DNA ends.
  • Only the endonuclease activity is required for the removal of DNA hairpins.
  • The MRX-Sae2 endonuclease introduces cleavages at varying distances from Spo11-blocked ends with decreasing efficiency.

Conclusions:

  • Sae2 stimulates the MRX endonuclease and 3'-5' exonuclease activities through distinct mechanisms mediated by Rad50.
  • These differential regulatory mechanisms ensure the diverse functions of the MRX-Sae2 nuclease at DNA ends during DSB repair.
  • The findings provide critical insights into the precise regulation of DNA repair pathways essential for maintaining genomic stability.

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