The role of SUMO E3 ligase RanBP2 in the DNA damage response

• Main Authors: Jyy-Shiuan Tu, 涂芷瑄
• Other Authors: Ching-Shyi Wu
• Format: Others
• Language: zh-TW
• Published: 2019
• Online Access: http://ndltd.ncl.edu.tw/handle/n7z2my

碩士 === 國立臺灣大學 === 藥理學研究所 === 107 === SUMOylation is one of post-translational modification, and SUMO conjugation to substrates occurs through a process involving an E1 activating enzyme, an E2 conjugating enzyme and an E3 protein ligase. Protein modification by SUMOylation can change the stability and activity of proteins and alter protein-protein interactions. Protein SUMOylation is important for several cellular processes, including transcription, DNA replication, chromosome segregation and DNA repair. In response to DNA damage, cells activate a series of mechanisms to protect genomic integrity termed the DNA damage response (DDR). Several types of post-translational modifications are known to regulate the DDR. Previous studies have shown that hundreds of the DDR proteins including RPA, ATRIP, and BRCA1 are SUMO conjugated in response to DNA damage. However, only few SUMO E3 ligases have been implicated in the DDR. It is not clear if other SUMO E3 ligases are also involved in the DDR. With a small scale siRNA screen, I find that RanBP2, a component of nuclear pore complex, is important for activation of the ATR-Chk1 signaling pathway. I find that knockdown of RanBP2 drastically reduces damage-induced phosphorylation of the ATR-Chk1 pathway but not the ATM-Chk2 pathway. Knockdown of RanBP2 does not significantly alter cell cycle progression, DNA synthesis, and formation of damage-induced DNA double-stranded breaks. Immunofluorescence imaging and Western blotting show that the foci formation and protein levels of γH2AX and RPA are not affected. Moreover, levels of several key factors of the ATR-Chk1 pathway remain unchanged after knockdown of RanBP2. Given the activation of ATR is the upstream event in the ATR-Chk1 pathway, I test whether RanBP2 interacts and regulates ATR directly. Immunoprecipitation of RanBP2 shows that ATR associates with RanBP2 during mitosis but not in interphase. The result suggests RanBP2 may affect the ATR-Chk1 pathway by interacting with ATR. However, it needs more work to test if RanBP2 affects the ATR-Chk1 pathway via its SUMO E3 function.