DNA-binding mechanism and evolution of replication protein A.

Area of Science:

• Structural biology
• Molecular biology
• Biochemistry

Background:

• Replication Protein A (RPA) is a crucial heterotrimeric protein for DNA replication, recombination, and repair.
• The structural and functional characteristics of RPA in Archaea remain largely uncharacterized.

Purpose of the Study:

• To elucidate the structure and function of RPA from the archaeon Pyrococcus abyssi.
• To investigate RPA's interactions with single-stranded DNA (ssDNA).
• To understand the evolutionary relationship of archaeal RPA to its eukaryotic counterparts.

Main Methods:

• Integrative structural biology (X-ray crystallography, cryo-electron microscopy)
• Biochemical assays
• Biophysical characterization

Main Results:

• Determined X-ray and cryo-EM structures of Pyrococcus abyssi RPA.
• Identified conserved trimerization core and ssDNA-binding interactions shared with eukaryotes.
• Discovered a novel helical domain, AROD (Acidic Rpa1 OB-binding Domain).
• Revealed the formation of an unexpected RPA tetrameric supercomplex in the absence of DNA.
• Demonstrated efficient ssDNA coating by the archaeal RPA tetramer.

Conclusions:

• Archaeal RPA shares fundamental structural and functional features with eukaryotic RPA.
• The novel AROD domain and tetrameric supercomplex represent unique archaeal adaptations.
• These findings provide crucial insights into the evolution of RPA as a primordial DNA replication factor.