Biochemical Characterization of Nucleostemin and Its Role in RAD51-Mediated DNA Repair
碩士 === 國立臺灣大學 === 生化科學研究所 === 105 === Cells constantly suffer various types of DNA damages including DNA double-strand breaks (DSBs). Notably, spontaneous DSBs are frequently generated in highly proliferating cells when replication forks were stalled or collapsed. Recent cell-based and animal studie...
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ndltd-TW-105NTU051030262019-05-15T23:39:45Z http://ndltd.ncl.edu.tw/handle/6axv4w Biochemical Characterization of Nucleostemin and Its Role in RAD51-Mediated DNA Repair 探討Nucleostemin生化特性及與重組酵素RAD51的交互作用 Bo-Ting Lin 林柏廷 碩士 國立臺灣大學 生化科學研究所 105 Cells constantly suffer various types of DNA damages including DNA double-strand breaks (DSBs). Notably, spontaneous DSBs are frequently generated in highly proliferating cells when replication forks were stalled or collapsed. Recent cell-based and animal studies have documented that nucleostemin (NS) participates in the DSB repair including replication-induced DSBs. NS belongs a G protein family and the expression is highly enriched in proliferating cells. Depletion of NS accumulates the spontaneous DSBs in S phase cells; conversely, overexpression of NS can significantly reduce the DSBs generated by stalled replication-fork upon the treatment of hydroxyurea. Moreover, NS forms a DSB-induced focus and interacts with RAD51, the key enzyme of homologous recombination-mediated DSB repair pathway. Interestingly, depletion of NS will significantly attenuate the recruitment of RAD51 to DSBs. Taken all results together, it is clear that nucleostemin participates in the RAD51-mediated recombination repair. However, it remains largely unknown regarding the NS biochemical characteristics and its functional interaction with RAD51-mediated DNA exchange, due to the hurdle of obtaining the NS recombinant proteins for the biochemical and functional analyses. Here, we have successfully established the expression and purification procedures of NS. Our biochemical analyses demonstrated that the purified monodispersed NS protein possesses a GTP binding ability. To our surprise, nucleostemin binds DNA with a preference for duplex DNA rather than single-strand DNA. Importantly, a direct protein-protein interaction between purified NS and RAD51 recombinase has been observed. Notably, we showed that the physical interaction is a species-specific since no interaction has been detected between mammalian NS and prokaryotic RecA recombinase. The functional significances of DNA binding and RAD51 interaction by NS on RAD51-mediated DSB repair will be examined in the near future. In summary, our NS purification system and the biochemical properties reported herein should expedite further mechanistic study regarding the mechanistic action of NS in RAD51-mediated DSB repair. 冀宏源 2017 學位論文 ; thesis 46 en_US |
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碩士 === 國立臺灣大學 === 生化科學研究所 === 105 === Cells constantly suffer various types of DNA damages including DNA double-strand breaks (DSBs). Notably, spontaneous DSBs are frequently generated in highly proliferating cells when replication forks were stalled or collapsed. Recent cell-based and animal studies have documented that nucleostemin (NS) participates in the DSB repair including replication-induced DSBs. NS belongs a G protein family and the expression is highly enriched in proliferating cells. Depletion of NS accumulates the spontaneous DSBs in S phase cells; conversely, overexpression of NS can significantly reduce the DSBs generated by stalled replication-fork upon the treatment of hydroxyurea. Moreover, NS forms a DSB-induced focus and interacts with RAD51, the key enzyme of homologous recombination-mediated DSB repair pathway. Interestingly, depletion of NS will significantly attenuate the recruitment of RAD51 to DSBs. Taken all results together, it is clear that nucleostemin participates in the RAD51-mediated recombination repair. However, it remains largely unknown regarding the NS biochemical characteristics and its functional interaction with RAD51-mediated DNA exchange, due to the hurdle of obtaining the NS recombinant proteins for the biochemical and functional analyses. Here, we have successfully established the expression and purification procedures of NS. Our biochemical analyses demonstrated that the purified monodispersed NS protein possesses a GTP binding ability. To our surprise, nucleostemin binds DNA with a preference for duplex DNA rather than single-strand DNA. Importantly, a direct protein-protein interaction between purified NS and RAD51 recombinase has been observed. Notably, we showed that the physical interaction is a species-specific since no interaction has been detected between mammalian NS and prokaryotic RecA recombinase. The functional significances of DNA binding and RAD51 interaction by NS on RAD51-mediated DSB repair will be examined in the near future. In summary, our NS purification system and the biochemical properties reported herein should expedite further mechanistic study regarding the mechanistic action of NS in RAD51-mediated DSB repair.
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author2 |
冀宏源 |
author_facet |
冀宏源 Bo-Ting Lin 林柏廷 |
author |
Bo-Ting Lin 林柏廷 |
spellingShingle |
Bo-Ting Lin 林柏廷 Biochemical Characterization of Nucleostemin and Its Role in RAD51-Mediated DNA Repair |
author_sort |
Bo-Ting Lin |
title |
Biochemical Characterization of Nucleostemin and Its Role in RAD51-Mediated DNA Repair |
title_short |
Biochemical Characterization of Nucleostemin and Its Role in RAD51-Mediated DNA Repair |
title_full |
Biochemical Characterization of Nucleostemin and Its Role in RAD51-Mediated DNA Repair |
title_fullStr |
Biochemical Characterization of Nucleostemin and Its Role in RAD51-Mediated DNA Repair |
title_full_unstemmed |
Biochemical Characterization of Nucleostemin and Its Role in RAD51-Mediated DNA Repair |
title_sort |
biochemical characterization of nucleostemin and its role in rad51-mediated dna repair |
publishDate |
2017 |
url |
http://ndltd.ncl.edu.tw/handle/6axv4w |
work_keys_str_mv |
AT botinglin biochemicalcharacterizationofnucleosteminanditsroleinrad51mediateddnarepair AT línbǎitíng biochemicalcharacterizationofnucleosteminanditsroleinrad51mediateddnarepair AT botinglin tàntǎonucleosteminshēnghuàtèxìngjíyǔzhòngzǔjiàosùrad51dejiāohùzuòyòng AT línbǎitíng tàntǎonucleosteminshēnghuàtèxìngjíyǔzhòngzǔjiàosùrad51dejiāohùzuòyòng |
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