Single-Cell Biophysics: Measurement, Modulation, and Modeling

Single-Cell Biophysics: Measurement, Modulation, and Modeling

Poster Abstracts

59-POS Board 30 Single-Molecule Biochemical Studies on the Stimulation Mechanism of SWI5-SFR1 Complex on RAD51 Presynaptic Filament Formation Chih-Hao Lu 1 , Hiroshi Iwasaki 2 , Peter Chi 3 , Hung-Wen Li 1 . 1 National Taiwan University, Taipei, Taiwan, 2 Tokyo Institute of Technology, Tokyo, Japan, 3 National Taiwan University, Taipei, Taiwan. Eukaryotic RAD51 protein is essential for DNA homologous recombinational repair of DNA damage. RAD51 recombinases assemble onto ssDNA to form a presynaptic filament, the required functional component for homology pairing and strand exchange reactions. This filament assembly is the committed step of homologous recombination and is subjected to regulation. Nucleation step is kinetically slow, and several accessory proteins have been identified to regulate RAD51 nucleation. SWI5-SFR1 (S5S1) is a heterodimeric accessory protein, and previous biochemical work showed that S5S1 interacts with RAD51, and stimulates RAD51-mediated homologous recombination. Our single-molecule tethered particle motion (TPM) experiments demonstrate that mouse S5S1 interacts with mouse RAD51 to form complex, and the mRAD51-S5S1 complex efficiently stimulates the nucleation step. We also showed that mS5S1 stimulates mRAD51 nucleation by (i) reducing mRAD51 seed size, (ii) increasing mRAD51 ssDNA affinity and (iii) stabilizing mRAD51 nucleus on ssDNA. While nucleation stimulation by S5S1 is absent in fission yeast (Schyzosaccharomyce pombe, Sp) system in our single-molecule work, SpS5S1 is shown to prevent SpRad51 disassembly. Different regulation strategies among species allow S5S1 to stabilize Rad51 filament efficiently.

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