A dynamic allosteric pathway underlies Rad50 ABC ATPase function in DNA repair

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• The Mre11-Rad50 protein complex initiates DNA double-strand break (DSB) repair.
• ATP binding to Rad50 induces structural changes vital for Mre11-Rad50 function.

Purpose of the Study:

• To elucidate the dynamic allosteric pathway within Rad50 using methyl-based NMR spectroscopy.
• To investigate the relationship between Rad50 structure, dynamics, and DNA repair activities.

Main Methods:

• Utilized methyl-based NMR spectroscopy on a series of Rad50 mutants.
• Analyzed changes in chemical environment and nanosecond timescale dynamics.
• Correlated chemical shift perturbations with Rad50 and Mre11 activities.

Main Results:

• Observed mutations altering the chemical environment and dynamics of Rad50 side chain methyl groups.
• Identified significant correlations between chemical shift perturbations and Mre11-Rad50 complex activities.
• Revealed an equilibrium between a ground state and an active, dimerization-competent state of Rad50.

Conclusions:

• The active Rad50 state exhibits altered local structure and dynamics, poised for ATP-induced dimerization and hydrolysis.
• A sparsely populated intermediate state of Rad50 is critical for Mre11-Rad50-directed DNA double-strand break repair.