Rtt105 regulates RPA function by configurationally stapling the flexible domains

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Replication Protein A (RPA) is crucial for DNA metabolism, binding single-stranded DNA (ssDNA) and interacting with numerous proteins.
• RPA's functions in DNA replication, repair, and recombination are tightly regulated.
• Rtt105 is a known regulator of RPA's nuclear localization.

Purpose of the Study:

• To elucidate the mechanism by which Rtt105 interacts with and regulates RPA.
• To understand Rtt105's role in preventing spurious RPA interactions.
• To investigate how Rtt105 modulates RPA-ssDNA filament formation and stability.

Main Methods:

• Biochemical assays to study Rtt105-RPA interactions.
• Analysis of RPA-ssDNA filament formation and stability.
• Investigation of Rtt105's effect on RPA-interacting protein binding.

Main Results:

• Rtt105 binds to RPA, acting as a conformational stabilizer.
• Rtt105 inhibits RPA binding to Rad52 in the absence of ssDNA, preventing off-target protein interactions.
• Rtt105 promotes RPA-ssDNA filament formation and stabilizes these filaments by inhibiting RPA exchange activity.

Conclusions:

• Rtt105 sequesters free RPA in the nucleus, preventing premature interactions with other proteins.
• Rtt105 plays a dual role: preventing inappropriate RPA binding and stabilizing RPA-ssDNA complexes at DNA lesion sites.
• These findings reveal a novel regulatory mechanism for RPA function in DNA metabolism.