Preparation of Nucleosome Core Particles Complexed with DNA Repair Factors for Cryo-Electron Microscopy Structural...

Area of Science:

• Molecular biology
• Structural biology
• Biochemistry

Background:

• DNA repair mechanisms within chromatin, the complex of DNA and proteins that forms chromosomes, remain largely uncharacterized.
• Nucleosomes, the fundamental units of chromatin, impede DNA repair enzyme activity compared to free DNA, necessitating structural investigation.
• The precise molecular interactions of base excision repair (BER) enzymes with nucleosomal DNA are not fully understood.

Purpose of the Study:

• To develop and present reproducible protocols for preparing nucleosomal DNA repair complexes for structural studies.
• To enable detailed structural determination of BER enzymes interacting with nucleosomes using cryo-electron microscopy (cryo-EM).

Main Methods:

• Development of two distinct methods for genetically fusing BER enzymes (Polymerase β and AP Endonuclease1).
• Preparation of nucleosomal complexes featuring fused BER enzymes bound to a single-nucleotide gap near the nucleosome entry-exit site.
• Optimization of sample preparation for high-quality cryo-electron microscopy grid vitrification via plunge freezing.

Main Results:

• Successful preparation of complexes containing fused BER enzymes and nucleosomes suitable for cryo-EM structural determination.
• Demonstration that the developed methods yield high-quality grids for structural analysis.
• Establishment of a foundational protocol adaptable for studying other chromatin-associated protein complexes.

Conclusions:

• The presented methods provide a viable approach for structural studies of DNA repair enzymes on nucleosomes.
• These protocols facilitate the investigation of how chromatin structure influences DNA repair mechanisms at a molecular level.
• This work serves as a basis for future structural studies of various chromatin-associated protein complexes, including transcription factors and chromatin modifiers.