Wrapping of single-stranded DNA by Replication Protein A and modulation through phosphorylation

Area of Science:

• Molecular Biology
• Biochemistry
• Structural Biology

Background:

• Replication Protein A (RPA) is crucial for shielding and processing single-stranded DNA (ssDNA) intermediates during DNA metabolism.
• Understanding RPA's interaction with ssDNA is key to elucidating its role in DNA repair and replication.
• Previous models proposed ssDNA stretching by RPA, but structural data suggested partial wrapping.

Approach:

• Utilized single-molecule Förster Resonance Energy Transfer (smFRET) and Double Electron-Electron Resonance (DEER) spectroscopy.
• Measured end-to-end distances of free ssDNA and RPA-ssDNA complexes.
• Investigated the impact of RPA70 phosphorylation on RPA-ssDNA complex structure.

Key Points:

• RPA binding causes only a small increase in ssDNA end-to-end distance, refuting linear stretching models.
• Findings support a model where ssDNA is partially wrapped around RPA's DNA-binding domains.
• Phosphorylation at Ser-384 of RPA70 facilitates remodeling of RPA domains, granting access to bound ssDNA.

Conclusions:

• Established a precise structural model for RPA-bound ssDNA, detailing partial wrapping.
• Demonstrated the role of RPA70 phosphorylation in regulating access to ssDNA.
• Provided critical insights into RPA's mechanism for remodeling ssDNA for downstream enzymatic pathways.