NBS1 promotes the endonuclease activity of the MRE11-RAD50 complex by sensing CtIP phosphorylation

Area of Science:

• Molecular Biology
• DNA Repair Mechanisms
• Eukaryotic DNA Metabolism

Background:

• DNA double-strand breaks (DSBs) are repaired via homologous recombination (HR).
• The initial step, DNA end resection, is performed by MRE11-RAD50-NBS1 (MRN) complex and CtIP.
• Human NBS1 plays a critical role in higher eukaryotes, suggesting complex regulatory mechanisms for the MRE11 nuclease.

Purpose of the Study:

• To elucidate the regulatory mechanisms of the MRE11 nuclease by NBS1 and CtIP.
• To investigate the role of NBS1 domains (FHA, BRCT) in sensing CtIP phosphorylation.
• To understand how NBS1 controls MRE11-RAD50 nuclease activity during DNA repair.

Main Methods:

• Utilized a reconstituted in vitro system with purified proteins.
• Investigated protein-protein interactions between NBS1, MRE11, and CtIP.
• Assessed MRE11-RAD50 nuclease activity under various conditions (with/without NBS1, phosphorylated/unphosphorylated CtIP).
• Performed in vivo experiments examining DNA end resection in NBS1 mutants.

Main Results:

• NBS1, via its FHA and BRCT domains, senses CtIP phosphorylation and activates MRE11-RAD50 nuclease.
• NBS1 directly interacts with MRE11 to enhance its nuclease activity.
• MRE11-RAD50 shows weaker nuclease activity without NBS1, requiring CtIP but not necessarily its phosphorylation.
• Two distinct modes of CtIP augmentation of MRE11 activity were identified: phosphorylation-dependent (via NBS1) and phosphorylation-independent (without NBS1).
• Limited DNA end resection occurs in vivo in the absence of NBS1 FHA and BRCT domains.

Conclusions:

• NBS1 acts as a crucial regulator, restricting MRE11-RAD50 nuclease activity to S-G2 phase when CtIP is highly phosphorylated.
• This study defines novel mechanisms controlling MRE11 nuclease activity, essential for accurate DNA repair.
• The findings highlight the complex regulation of DNA end resection in higher eukaryotes.