Constructing arrays of nucleosome positioning sequences using Gibson Assembly for single-molecule studies

Area of Science:

• Molecular Biology
• Genomics
• Biophysics

Background:

• Nucleosomes are fundamental to chromatin structure and regulate eukaryotic genome accessibility.
• Nucleosome involvement is critical in DNA transcription, replication, and repair processes.
• Single-molecule techniques are vital for studying nucleosome-DNA interactions and protein binding.

Purpose of the Study:

• To present a novel and efficient Gibson Assembly method for creating DNA molecules with defined nucleosome positioning sequences.
• To facilitate the generation of controlled nucleosome arrays for advanced single-molecule studies.
• To investigate nucleosome unwrapping and histone binding dynamics under mechanical tension.

Main Methods:

• Gibson Assembly cloning was utilized to synthesize DNA molecules with specific arrays of the '601' nucleosome positioning sequence.
• Reconstitution of nucleosome arrays on synthesized DNA molecules.
• Dual-trap optical tweezers and fluorescence microscopy were employed to apply tension and monitor nucleosome behavior.

Main Results:

• A novel, efficient method for generating DNA with defined nucleosome arrays was established.
• Nucleosomes were observed to unwind at approximately 20 pN of tension.
• Histone proteins, specifically histone H3, were found to remain associated with DNA at tensions exceeding 60 pN.

Conclusions:

• The developed Gibson Assembly method offers improved accessibility and control for creating nucleosome arrays.
• Nucleosome unwrapping does not necessarily lead to complete histone dissociation from DNA.
• These findings provide crucial insights into the mechanical stability and structural dynamics of chromatin at the single-molecule level.