Mus81-Mms4 endonuclease is an Esc2-STUbL-Cullin8 mitotic substrate impacting on genome integrity

Area of Science:

• Molecular Biology
• Cell Cycle Regulation
• DNA Repair

Background:

• The Mus81-Mms4 nuclease resolves DNA recombination structures during the G2/M phase, a process requiring Mms4 phosphorylation.
• The fate of phosphorylated Mms4 after its activation and function remains unclear.

Purpose of the Study:

• To investigate the post-activation fate of phosphorylated Mms4.
• To understand the regulatory mechanisms controlling Mms4 levels and its impact on genome stability.

Main Methods:

• SUMOylation and ubiquitylation assays to detect Mms4 modification.
• Proteasome degradation assays.
• Chromatin immunoprecipitation to assess Mms4 localization.
• Analysis of DNA damage checkpoint activation and genome integrity in wild-type and mutant strains.

Main Results:

• Phosphorylated Mms4 undergoes (poly)SUMOylation and ubiquitylation, targeting it for proteasome degradation.
• Mms4 is a substrate for the Esc2-STUbL-Cul8 ubiquitin ligase complex.
• Loss of Esc2-STUbL-Cul8 activity leads to Mms4 accumulation on chromatin, causing aberrant processing of recombination intermediates and delayed DNA damage checkpoint activation.
• Mutants stabilizing phosphorylated Mms4 compromise genome integrity.

Conclusions:

• Mms4 is regulated by proteasomal degradation, a process crucial for resetting its activity between cell cycles.
• The Esc2-STUbL-Cul8 complex plays a key role in replication protection by ensuring timely Mms4 turnover.
• Proper regulation of phosphorylated Mms4 is essential for maintaining genome stability.