Structure guided functional analysis of the S. cerevisiae Mre11 complex

Area of Science:

• Molecular Biology
• Structural Biology
• Biochemistry

Background:

• The Mre11 complex, comprising Mre11, Rad50, and Nbs1/Xrs2, is essential in eukaryotes for DNA double-strand break (DSB) repair.
• Mre11 and Rad50 are highly conserved across life, while Nbs1/Xrs2 is eukaryotic-specific.
• The complex plays a crucial role in detecting DNA damage and activating signaling pathways.

Purpose of the Study:

• To determine the cryo-electron microscopy (cryo-EM) structure of the Saccharomyces cerevisiae Mre11-Rad50 complex bound to double-stranded DNA (dsDNA).
• To elucidate the molecular mechanisms underlying complex assembly, DNA binding, and the functional roles of conserved residues.
• To investigate the influence of the Rad50 coiled-coil domain on ATP hydrolysis.

Main Methods:

• Cryo-electron microscopy (cryo-EM) to obtain a 3.2 Å resolution structure.
• Biochemical assays and mutational analyses to investigate protein interfaces and DNA binding.
• Structural analysis to identify conserved residues critical for complex function.

Main Results:

• A high-resolution cryo-EM structure of the Mre11-Rad50-dsDNA complex was obtained.
• Identified conserved residues in Mre11 and Rad50 essential for complex assembly and DNA interaction.
• Demonstrated that the Rad50 coiled-coil domain allosterically affects ATP hydrolysis distant from the catalytic site.

Conclusions:

• The determined structure provides a molecular basis for understanding Mre11-Rad50 complex function in DNA repair.
• Conserved residues are key for the structural integrity and DNA-binding capabilities of the complex.
• Rad50's coiled-coil domain plays a regulatory role in the complex's enzymatic activity.