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Single-Cell Biophysics: Measurement, Modulation, and Modeling
Poster Abstracts
74
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.