The mechanistic study of SWI5-SFR1 complex on RAD51-mediated DNA repair
Date Issued
2016
Date
2016
Author(s)
Su, Guan-Chin
Abstract
Homologous recombination (HR) mediated by RAD51 recombinase eliminates DNA double-strand breaks in the genome. RAD51 forms a nucleoprotein filament on single-stranded DNA (ssDNA), termed the presynaptic filament, to initiate homologous recombination. Cytological studies in mammal indicate that SWI5 and SFR1 form a complex and participate in RAD51-mediated recombination repair. To decipher the mechanistic role of mammal SWI5-SFR1 complex in RAD51-mediated HR, we established the expression and the purification procedure to obtain mouse SWI5-SFR1 protein complex. Our biochemical study showed that SWI5-SFR1 complex stimulates homologous DNA pairing by RAD51 and stabilizes the RAD51 presynaptic filament, demonstrating that the stimulation of RAD51 activity stems from the stabilization of RAD51 filament by SWI5-SFR1 complex. RAD51 is an ssDNA dependent ATPase. ATP binding promotes the formation of a functional RAD51 nucleoprotein filament and DNA stand exchange activity. However, owing to ATP hydrolysis and slow dissociation rate of ADP, active RAD51 filament is converted into an inactive RAD51-ADP-ssDNA filament. We further documented that SWI5-SFR1 acts by facilitating the release of ADP from the RAD51 presynaptic filament, indicating that SWI5-SFR1 helps maintain RAD51 presynaptic filament in its active ATP bound form. Furthermore, we determined the interaction mode and region between SWI5-SFR1 and RAD51. We found that SWI5-SFR1 preferentially interacts with oligomeric form of RAD51. Importantly, the residue F83 and L85 in C-terminal SWI5 of SWI5-SFR1 complex is essential for the interaction of RAD51. The interaction of SWI5-SFR1 to RAD51 is indispensable for stimulation of RAD51 activity. Our results thus provide the insight for the action mechanism of RAD51-mediated DNA exchange by SWI5-SFR1 complex.
Subjects
Homologous recombination
DNA double-strand breaks
RAD51 presynaptic filament
ATP hydrolysis
ADP release
Type
thesis
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