Abstract
SWI5-SFR1 protein complex (S5S1) has been proven as an accessory factor of homologous recombination repair, which can restore the DNA double strands break (DSB). When DNA double strands break (DSB) happen, RAD51 recombinases (RAD51) as a key protein form a presynaptic filament via binding to a single-stranded DNA. Then, ATP would bind to RAD51 to stabilize the RAD51-DNA complex and induce single-stranded DNA exchange. Due to the hydrolytic activity of RAD51, ATP would be hydrolyzed to ADP and the activity of RAD51-DNA complex will decrease, and S5S1 can promote the ADP releasing rate by interaction between RAD51. Recently, two active sites, F83 and L85, on the C-terminus of SWI5 (S5) have been reported to participate in the interaction between S5S1 and RAD51. In order to investigate their interaction, we use molecular modeling with the template “S5S1c from yeast (which has a known crystal structure, PDB code: 3VIQ)” to construct the model, we found that S5F83 and S5L85 are located in the hydrophobic core in the ending of S5 and faced inside the core. Hence, we speculate the two positions would expose and attach to RAD51 when the bundle structure on the S5 C-terminal alpha-helix was separated. To validate our hypothesis, first, we fix the C-terminal loop by creating the disulfide bond S5D89C-S1C53 on the complex. In addition, to prevent the other disulfide bond take place in the molecular, we also replaced the S1C59 to Ser. Next, we compared the secondary and tertiary structure by circular dichroism (CD) and small-angle X-ray scattering (SAXS). CD analysis showed that both S5S1c and S5D89CS1cC59S presented mainly alpha-helical structure, and their secondary structure compositions are similar by analyzed on the DichroWEB website. For SAXS experiment, we determined the structure characteristic of S5S1c and mutant, and constructed the ab initio model of them. The results showed no huge difference between S5S1c and mutant. Finally, the binding ability was determined by pull-down assay. We compared S5S1c, loop-fixed and unfixed S5D89CS1cC59S (without and with 2-ME treatment) The results showed that, compare to S5S1c, loop-fixed S5D89CS1cC59S cannot interact to RAD51, and it restored partially binding ability when freed the loop which indicate S5F83 and S5L85 could expose and bind to RAD51. In conclusion, we speculate the opening of S5 C-terminal loop is essential when S5S1c interact to RAD51. Our study provide information regarding to S5S1c binding to RAD51.