Ring-shaped Rad51 paralog protein complexes bind Holliday junctions and replication forks as visualized by electron...

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Mammals possess five Rad51 paralogs that assemble into two distinct protein complexes: BCDX2 (Rad51B-Rad51C-Rad51D-Xrcc2) and CX3 (Rad51C-Xrcc3).
• These complexes play critical roles in DNA replication and recombination repair pathways.
• Understanding their structure and DNA-binding preferences is essential for elucidating their function.

Purpose of the Study:

• To investigate the DNA binding preferences and structural characteristics of human BCDX2 and CX3 complexes.
• To visualize the interaction of these complexes with DNA structures like replication forks and Holliday junctions.
• To determine the overall architecture of the BCDX2 and CX3 complexes.

Main Methods:

• Co-expression and purification of human BCDX2 and CX3 complexes in insect cells.
• Transmission electron microscopy (TEM) for structural visualization.
• Analysis of complex binding to DNA templates containing replication forks and Holliday junctions.

Main Results:

• Both BCDX2 and CX3 complexes exhibit high specificity for binding to DNA junctions.
• TEM revealed that the free and DNA-bound complexes adopt a multimeric ring structure.
• The ring structure features subunits arranged in a flat disc around a central channel.

Conclusions:

• This study provides the first electron microscopy visualization of BCDX2 and CX3 binding to Holliday junctions and forked DNA structures.
• The findings suggest that BCDX2 and CX3 complexes form ring-shaped structures involved in DNA repair.
• The specific binding to DNA junctions highlights their potential role in processing these critical DNA structures during replication and recombination.