Smc5/6 uses its head-NSE module to preferentially associate with ssDNA gaps and ss-dsDNA junctions

Area of Science:

• Molecular Biology
• Genetics
• Biophysics

Background:

• Structural Maintenance of Chromosomes (SMC) complexes are vital for genome stability.
• While cohesin and condensin organize genome architecture, Smc5/6 specifically facilitates DNA replication and repair.
• Previous studies used fluorescence microscopy to infer Smc5/6 interactions with replication and repair intermediates.

Purpose of the Study:

• To elucidate the high-resolution structure and DNA-binding mechanisms of the budding yeast Smc5/6 complex.
• To investigate the interaction of Smc5/6 with single-stranded DNA (ssDNA) gaps using atomic force microscopy (AFM).

Main Methods:

• Atomic force microscopy (AFM) was utilized to visualize the budding yeast Smc5/6 complex at nanometer resolution.
• AFM was employed to study the interaction of Smc5/6 with DNA structures, including ssDNA gaps and double-stranded DNA (dsDNA).
• Mutational analysis of DNA-binding residues within the Smc5/6 complex was performed.

Main Results:

• The Smc5/6 complex exhibits a 'bead-on-stick' conformation, with distinct head-NSE and hinge domains.
• Smc5/6 primarily binds to dsDNA via its head-NSE module, with mutations shifting binding to the hinge domain.
• The complex preferentially associates with ssDNA gaps and ss-dsDNA junctions over dsDNA, mediated mainly by the head-NSE module.

Conclusions:

• AFM imaging provides a high-resolution structural and mechanistic understanding of Smc5/6-DNA interactions.
• Smc5/6's preference for ssDNA gaps and junctions highlights its direct role in processing replication and repair intermediates.
• These findings offer a framework for comprehending Smc5/6's essential functions in maintaining genome integrity.