Partial wrapping of single-stranded DNA by replication protein A and modulation through phosphorylation

Area of Science:

• Molecular Biology
• Structural Biology
• Biochemistry

Background:

• Replication protein A (RPA) is crucial for shielding and directing single-stranded DNA (ssDNA) intermediates during DNA metabolism.
• Understanding RPA's mechanism requires knowing the structural conformation of RPA-bound ssDNA.

Purpose of the Study:

• To reconcile conflicting models of ssDNA conformation upon RPA binding.
• To determine the precise structural model of ssDNA when bound by RPA.
• To investigate the role of RPA70 phosphorylation in regulating ssDNA accessibility.

Main Methods:

• Single-molecule Förster Resonance Energy Transfer (smFRET) spectroscopy.
• Double Electron-Electron Resonance (DEER) spectroscopy to measure end-to-end distances of ssDNA and RPA-ssDNA complexes.

Main Results:

• RPA binding causes only a small increase in ssDNA end-to-end distance, supporting a partial wrapping model over linear stretching.
• Structural data reveals ssDNA is partially wrapped around RPA's DNA-binding domains.
• Phosphorylation at Ser-384 of RPA70 remodels RPA domains, granting access to the wrapped ssDNA.

Conclusions:

• A precise structural model for RPA-bound ssDNA is established, showing partial wrapping.
• RPA's remodeling of ssDNA is key for facilitating downstream DNA metabolic pathways.
• RPA70 phosphorylation plays a critical role in regulating RPA function by controlling ssDNA accessibility.