Determinants of replication protein A subunit interactions revealed using a phosphomimetic peptide

Area of Science:

• Molecular biology
• Biochemistry
• Structural biology

Background:

• Replication protein A (RPA) is crucial for DNA metabolism, involving subunits RPA70, RPA32, and RPA14.
• Phosphorylation of RPA32 regulates DNA replication and damage response, influencing RPA's interaction with partners like Bloom syndrome helicase (BLM).
• Structural determinants of the phosphorylated RPA32-RPA70 interaction remain unclear, hindering understanding of DNA resection regulation.

Purpose of the Study:

• To elucidate the molecular details and structural determinants of the interaction between RPA70 and a phosphorylated RPA32 mimic (pmRPA32).
• To understand how phosphorylation of RPA32 modulates its binding to RPA70 and influences DNA resection inhibition.

Main Methods:

• Utilized fluorescence polarization and Nuclear Magnetic Resonance (NMR) spectroscopy to analyze RPA70-pmRPA32 interactions.
• Employed alanine scanning mutagenesis on pmRPA32 to identify key binding residues.
• Performed molecular docking simulations to predict the complex orientation.

Main Results:

• The N-terminal domain of RPA70 (RPA70N) specifically binds pmRPA32, but not unphosphorylated RPA32.
• NMR data indicated that RPA70N binds pmRPA32 via a basic cleft region.
• At least six negatively charged residues on pmRPA32 are essential for RPA70N binding.
• Mutagenesis and docking simulations identified potential contact points and suggested the orientation of the pmRPA32-RPA70N complex.

Conclusions:

• Detailed structural features of the RPA70-RPA32 domain-domain interaction upon RPA32 phosphorylation have been revealed.
• Phosphorylation of RPA32 significantly alters its binding to RPA70, providing insights into the regulation of transient protein interactions in DNA processing pathways.
• This work clarifies how phosphorylation controls RPA interactions, impacting DNA resection inhibition.