A structural and dynamic model for the assembly of Replication Protein A on single-stranded DNA

Area of Science:

• Molecular Biology
• Structural Biology
• Biochemistry

Background:

• Replication Protein A (RPA) is crucial for eukaryotic DNA replication and repair.
• RPA binds single-stranded DNA (ssDNA) to protect it and recruit other proteins.
• The exact structure and assembly mechanism of RPA on ssDNA are not fully understood.

Purpose of the Study:

• To elucidate the molecular architecture of the RPA trimerization core bound to ssDNA.
• To investigate the dynamic rearrangements of RPA's DNA binding domains (DBDs) during assembly.
• To understand how RPA binding is regulated, particularly by phosphorylation.

Main Methods:

• Cryo-electron microscopy (cryo-EM) for high-resolution 3D reconstruction.
• Fluorescence Resonance Energy Transfer (FRET) for solution-based dynamic studies.
• Structural modeling to propose assembly mechanisms.

Main Results:

• A 3D reconstruction of the RPA trimerization core bound to ssDNA at ~4.7 Å resolution was obtained.
• A dimeric RPA assembly on ssDNA was visualized.
• FRET studies demonstrated dynamic DBD rearrangements during RPA binding, regulated by RPA70 S178 phosphorylation.

Conclusions:

• Dynamic DBDs are key to the cooperative assembly of multiple RPA molecules on long ssDNA.
• Phosphorylation of RPA70 at S178 plays a regulatory role in RPA's DNA binding activity.
• The findings provide a structural model for RPA's function in nucleic acid transactions.