The mechanism of SWI5-SFR1 and RAD51AP1 stimulating RAD51-mediated recombination

• Main Authors: Chia-Yu Liao, 廖嘉妤
• Other Authors: Hung-Yuan Chi
• Format: Others
• Language: en_US
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/35158559157847550335

碩士 === 國立臺灣大學 === 生化科學研究所 === 101 === Homologous recombination (HR) is the major pathway to repair DNA double-strand breaks (DSBs). Dysfunction of HR leads to genome instability and even cancer formation. When a double-stranded DNA (dsDNA) breaks, the break ends undergo a resection process to generate a single-stranded DNA (ssDNA), and then RAD51 recombinase assembles on ssDNA to form a nucleoprotein filament, which is called presynaptic filament. The presynaptic filament captures and invades in dsDNA to search the homologous DNA. Finally, the ssDNA exchanges with their homologous DNA within dsDNA. During this HR reaction, the activity of RAD51 is regulated by accessory factors, including SWI5-SFR1 and RAD51AP1. Previous cell-based studies showed that knockdown SWI5-SFR1 or RAD51AP1 in mammalian cells results in increased sensitivity to DNA damaging agents. It raises an intriguing question how SWI5-SFR1 and RAD51AP1 mechanistically co-regulate Rad51 recombinase activity during HR. To address this question, we use purified proteins and in vitro reconstitution system to study their mechanistic actions in recombination reaction. Our results indicate that mouse SWI5-SFR1 and RAD51AP1 exhibit a synergistic effect on RAD51-mediated strand exchange. Moreover, the synergistic effect not only requires SWI5-SFR1 forming complex interacting with RAD51, but also requires RAD51AP1 binding DNA and RAD51. Furthermore, we found that SWI5-SFR1 can’t associate with RAD51AP1 directly, but SWI5-SFR1, RAD51AP1 and RAD51 form a ternary complex. Taken together, our findings suggest that the synergistic effect stems from the stimulation of SWI5-SFR1 and RAD51AP1 individually, and provide a mechanical insight of accessory factors in homologous recombination.