MksEF Accessory Proteins Inhibit MksB ATPase Activity and Modulate DNA Substrate Binding

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• Chromosome organization and segregation are vital cellular processes.
• Mechanisms of bacterial nucleoid organization are not fully understood.
• Structural Maintenance of Chromosomes (SMC) complexes play key roles in genome management.

Purpose of the Study:

• Investigate the function of the MksBEF SMC complex in *Mycobacterium smegmatis*.
• Elucidate the role of MksB, MksE, and MksF in chromosome organization.
• Understand the regulatory mechanisms of this alternative SMC complex.

Main Methods:

• Reconstitution of the MksBEF complex.
• Determination of complex stoichiometry (MksB₂E₄F₂) using gel filtration and ultracentrifugation.
• DNA-binding assays (gel shift, isothermal titration calorimetry) and fluorescence spectroscopy (ANS) to study protein-DNA interactions and structural changes.

Main Results:

• MksB binds DNA and is crucial for local chromosome organization.
• MksE and MksF enhance MksB's DNA-binding affinity through direct interaction.
• DNA binding induces structural rearrangements in MksB and the MksBEF complex.
• MksEF suppresses MksB's ATPase activity, a novel regulatory mechanism.

Conclusions:

• The MksBEF complex represents an alternative SMC system with unique regulatory functions.
• MksE and MksF modulate MksB activity, impacting DNA binding and ATPase function.
• Findings advance the understanding of chromosome dynamics and organization in mycobacteria.