ATP half-sites in RadA and RAD51 recombinases bind nucleotides

Area of Science:

• Biochemistry
• Molecular Biology
• Genetics

Background:

• Homologous recombination is a critical DNA repair pathway for double-strand breaks.
• Recombinases, such as archaeal RadA and human RAD51, are central to this process.
• These proteins form nucleoprotein filaments on single-stranded DNA to facilitate ATP-dependent repair.

Purpose of the Study:

• To investigate the nucleotide-binding properties of monomeric RadA and RAD51.
• To elucidate the structural basis of nucleotide binding in RadA through crystallography.

Main Methods:

• Expression and purification of truncated, monomeric Pyrococcus furiosus RadA and monomerized human RAD51.
• High-affinity nucleotide binding assays.
• X-ray crystallography of apo and nucleotide-bound monomeric RadA.

Main Results:

• Monomeric RadA and RAD51 exhibit high-affinity binding to ATP and other nucleotides.
• Crystal structures reveal tight phosphate binding but a poorly defined base-binding surface in monomeric RadA.
• The bound nucleotide in monomers may play a structural role in filament formation.

Conclusions:

• Monomeric RadA likely binds nucleotides constitutively within the cell.
• Nucleotide binding in monomers could be important for initiating RadA filament assembly.
• Understanding these mechanisms provides insight into DNA repair pathways.