探討Nucleostemin生化特性及與重組酵素RAD51的交互作用

Biochemical Characterization of Nucleostemin and Its Role in RAD51-Mediated DNA Repair

10.6342/NTU201700903

林柏廷

Nucleostemin ； 雙股去氧核醣核酸斷裂 ； 同源重組 ； RAD51重組酵素 ； 三磷酸鳥苷結合蛋白 ； Nucleostemin ； Double-strand breaks ； Homologous recombination ； RAD51 recombinase ； GTP binding protein

臺灣大學生化科學研究所學位論文

2017年

碩士

冀宏源

英文

染色體複製(replication)是細胞增生的重要步驟，在具有高度增生能力的細胞中，頻繁的複製過程容易造成因複製叉停滯(stalled replication fork)，或是複製叉崩塌(collapse of replication fork)而形成的雙股去氧核醣核酸斷裂斷裂(DNA double-strand breaks)，進而導致基因的不穩定。在過去研究發現，Nucleostemin (NS)參與在複製所產生雙股去氧核醣核酸斷裂的修復機制中。NS是屬於三磷酸鳥苷結合蛋白並在高度增生細胞中大量表達，目前已知若細胞中缺乏NS，複製過程將累積大量自發性的雙股去氧核醣核酸斷裂；另一方面，過度表達NS則能夠減少在複製機制中hydroxyurea所引發的雙股去氧核醣核酸斷裂。值得注意的是在近期的研究報導，NS與修復雙股去氧核醣核酸斷裂的重組酵素RAD51同時存在於相同的免疫沉澱體；且NS的缺少更進一步地影響RAD51在斷裂染色體上存在的數量，顯示NS參與在RAD51相關的修復機制之中。然而過去由於純化NS蛋白相當困難，使其本身蛋白生化特性以及與RAD51之間交互作用以完成修復的詳細機制仍有許多未知。在我們的研究中，我們建立了表達與純化NS重組蛋白的系統，並且成功地得到了高純度的NS蛋白質，利用NS重組蛋白，我們證明了NS能夠與三磷酸鳥苷結合。在凝膠遷移實驗中發現，NS具有去氧核醣核酸的結合能力並且傾向與雙股去氧核醣核酸結合。根據親和性沉降測試，我們發現哺乳類的NS與RAD51有直接的蛋白質交互作用，但不會與原核生物的重組酵素RecA進行交互作用。然而NS的去氧核醣核酸結合能力以及與RAD51交互作用對於NS所參與的去氧核醣核酸修復機制為何，仍然需要進一步分析。

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.

1. 1. Baddoo, M., Hill, K., Wilkinson, R., Gaupp, D., Hughes, C., Kopen, G.C., and Phinney, D.G. (2003). Characterization of mesenchymal stem cells isolated from murine bone marrow by negative selection. Journal of Cellular Biochemistry 89, 1235-1249.
   連結：
2. 2. Bassler, J., Kallas, M., and Hurt, E. (2006). The NUG1 GTPase Reveals an N-terminal RNA-binding Domain That Is Essential for Association with 60 S Pre-ribosomal Particles. Journal of Biological Chemistry 281, 24737-24744.
   連結：
3. 3. Beekman, C., Nichane, M., De Clercq, S., Maetens, M., Floss, T., Wurst, W., Bellefroid, E., and Marine, J.-C. (2006). Evolutionarily Conserved Role of Nucleostemin: Controlling Proliferation of Stem/Progenitor Cells during Early Vertebrate Development. Molecular and Cellular Biology 26, 9291-9301.
   連結：
4. 4. Bishop, D.K., Park, D., Xu, L., and Kleckner, N. (1992). DMC1: A meiosis-specific yeast homolog of E. coli recA required for recombination, synaptonemal complex formation, and cell cycle progression. Cell 69, 439-456.
   連結：
5. 5. Du, X., Rao, M.R.K.S., Chen, X.Q., Wu, W., Mahalingam, S., and Balasundaram, D. (2006). The Homologous Putative GTPases Grn1p from Fission Yeast and the Human GNL3L Are Required for Growth and Play a Role in Processing of Nucleolar Pre-rRNA. Molecular Biology of the Cell 17, 460-474.
   連結：
6. 7. Hashimoto, Y., Chaudhuri, A.R., Lopes, M., and Costanzo, V. (2010). Rad51 protects nascent DNA from Mre11-dependent degradation and promotes continuous DNA synthesis. Nat Struct Mol Biol 17, 1305-1311.
   連結：
7. 8. Hsu, J.K., Lin, T., and Tsai, R.Y.L. (2012). Nucleostemin prevents telomere damage by promoting PML-IV recruitment to SUMOylated TRF1. The Journal of cell biology 197, 613-624.
   連結：
8. 9. Jeon, Y., Park, Y.-J., Cho, H.K., Jung, H.J., Ahn, T.-K., Kang, H., and Pai, H.-S. (2015). The nucleolar GTPase nucleostemin-like 1 plays a role in plant growth and senescence by modulating ribosome biogenesis. Journal of Experimental Botany 66, 6297-6310.
   連結：
9. 10. Kafienah, W., Mistry, S., Williams, C., and Hollander, A.P. (2006). Nucleostemin Is a Marker of Proliferating Stromal Stem Cells in Adult Human Bone Marrow. STEM CELLS 24, 1113-1120.
   連結：
10. 11. Kudron, M.M., and Reinke, V. (2008). C. elegans Nucleostemin Is Required for Larval Growth and Germline Stem Cell Division. PLOS Genetics 4, e1000181.
    連結：
11. 12. Lin, T., Ibrahim, W., Peng, C.-Y., Finegold, M.J., and Tsai, R.Y.L. (2013). A novel role of nucleostemin in maintaining the genome integrity of dividing hepatocytes during mouse liver development and regeneration. Hepatology 58, 2176-2187.
    連結：
12. 13. Lin, T., Meng, L., Lin, T.-C., Wu, L.J., Pederson, T., and Tsai, R.Y.L. (2014). Nucleostemin and GNL3L exercise distinct functions in genome protection and ribosome synthesis, respectively. Journal of cell science 127, 2302-2312.
    連結：
13. 14. Lin, T., Meng, L., Li, Y., and Tsai, R.Y.L. (2010). Tumor-Initiating Function of Nucleostemin-Enriched Mammary Tumor Cells. Cancer Research 70, 9444-9452.
    連結：
14. 15. Lin, Z., Kong, H., Nei, M., and Ma, H. (2006). Origins and evolution of the recA/RAD51 gene family: Evidence for ancient gene duplication and endosymbiotic gene transfer. Proceedings of the National Academy of Sciences 103, 10328-10333.
    連結：
15. 16. Mazina, O.M., Rossi, M.J., Thomaä, N.H., and Mazin, A.V. (2007). Interactions of Human Rad54 Protein with Branched DNA Molecules. Journal of Biological Chemistry 282, 21068-21080.
    連結：
16. 17. Meng, L., Lin, T., Peng, G., Hsu, J.K., Lee, S., Lin, S.-Y., and Tsai, R.Y.L. (2013). Nucleostemin deletion reveals an essential mechanism that maintains the genomic stability of stem and progenitor cells. Proceedings of the National Academy of Sciences of the United States of America 110, 11415-11420.
    連結：
17. 18. Meng, L., Yasumoto, H., and Tsai, R.Y.L. (2006). Multiple controls regulate nucleostemin partitioning between nucleolus and nucleoplasm. Journal of cell science 119, 5124-5136.
    連結：
18. 20. Petermann, E., Orta, M.L., Issaeva, N., Schultz, N., and Helleday, T. (2010). Hydroxyurea-Stalled Replication Forks Become Progressively Inactivated and Require Two Different RAD51-Mediated Pathways for Restart and Repair. Molecular Cell 37, 492-502.
    連結：
19. 21. Politz, J.C.R., Polena, I., Trask, I., Bazett-Jones, D.P., and Pederson, T. (2005). A Nonribosomal Landscape in the Nucleolus Revealed by the Stem Cell Protein Nucleostemin. Molecular Biology of the Cell 16, 3401-3410.
    連結：
20. 22. Qu, J., and Bishop, J.M. (2012). Nucleostemin maintains self-renewal of embryonic stem cells and promotes reprogramming of somatic cells to pluripotency. The Journal of cell biology 197, 731-745.
    連結：
21. 23. Rosby, R., Cui, Z., Rogers, E., deLivron, M.A., Robinson, V.L., and DiMario, P.J. (2009). Knockdown of the Drosophila GTPase Nucleostemin 1 Impairs Large Ribosomal Subunit Biogenesis, Cell Growth, and Midgut Precursor Cell Maintenance. Molecular Biology of the Cell 20, 4424-4434.
    連結：
22. 24. San Filippo, J., Sung, P., and Klein, H. (2008). Mechanism of eukaryotic homologous recombination. Annu Rev Biochem 77, 229-257.
    連結：
23. 25. Schlacher, K., Christ, N., Siaud, N., Egashira, A., Wu, H., and Jasin, M. (2011). Double-Strand Break Repair-Independent Role for BRCA2 in Blocking Stalled Replication Fork Degradation by MRE11. Cell 145, 529-542.
    連結：
24. 26. Schlacher, K., Wu, H., and Jasin, M. (2012). A Distinct Replication Fork Protection Pathway Connects Fanconi Anemia Tumor Suppressors to RAD51-BRCA1/2. Cancer Cell 22, 106-116.
    連結：
25. 27. Sharan, S.K., Morimatsu, M., Albrecht, U., Lim, D.-S., Regel, E., Dinh, C., Sands, A., Eichele, G., Hasty, P., and Bradley, A. (1997). Embryonic lethality and radiation hypersensitivity mediated by Rad51 in mice lacking Brca2. Nature 386, 804-810.
    連結：
26. 28. Sharma, V., Khurana, S., Kubben, N., Abdelmohsen, K., Oberdoerffer, P., Gorospe, M., and Misteli, T. (2015). A BRCA1‐interacting lncRNA regulates homologous recombination. EMBO reports 16, 1520-1534.
    連結：
27. 30. Sung, P., and Klein, H. (2006). Mechanism of homologous recombination: mediators and helicases take on regulatory functions. Nat Rev Mol Cell Biol 7, 739-750.
    連結：
28. 31. Tsai, S.-P., Su, G.-C., Lin, S.-W., Chung, C.-I., Xue, X., Dunlop, M.H., Akamatsu, Y., Jasin, M., Sung, P., and Chi, P. (2012). Rad51 presynaptic filament stabilization function of the mouse Swi5–Sfr1 heterodimeric complex. Nucleic Acids Research 40, 6558-6569.
    連結：
29. 32. Tsai, R.Y.L. (2011). New Frontiers in Nucleolar Research: Nucleostemin and Related Proteins. In The Nucleolus, M.O.J. Olson, ed. (New York, NY: Springer New York), pp. 301-320.
    連結：
30. 33. Tsai, R.Y.L. (2014). Turning a new page on nucleostemin and self-renewal. Journal of cell science 127, 3885-3891.
    連結：
31. 34. Tsai, R.Y.L., and McKay, R.D.G. (2002). A nucleolar mechanism controlling cell proliferation in stem cells and cancer cells. Genes & Development 16, 2991-3003.
    連結：
32. 35. Tsai, R.Y.L., and McKay, R.D.G. (2005). A multistep, GTP-driven mechanism controlling the dynamic cycling of nucleostemin. The Journal of cell biology 168, 179-184.
    連結：
33. 36. Tsuzuki, T., Fujii, Y., Sakumi, K., Tominaga, Y., Nakao, K., Sekiguchi, M., Matsushiro, A., Yoshimura, Y., and MoritaT (1996). Targeted disruption of the Rad51 gene leads to lethality in embryonic mice. Proceedings of the National Academy of Sciences 93, 6236-6240.
    連結：
34. 37. Wang, Anderson T., Kim, T., Wagner, John E., Conti, Brooke A., Lach, Francis P., Huang, Athena L., Molina, H., Sanborn, Erica M., Zierhut, H., Cornes, Belinda K., et al. (2015). A Dominant Mutation in Human RAD51 Reveals Its Function in DNA Interstrand Crosslink Repair Independent of Homologous Recombination. Molecular Cell 59, 478-490.
    連結：
35. 38. Wittinghofer, A., and Vetter, I.R. (2011). Structure-Function Relationships of the G Domain, a Canonical Switch Motif. Annual Review of Biochemistry 80, 943-971.
    連結：
36. 39. Zhang, B., Zhang, Y., Wang, Z.-x., and Zheng, Y. (2000). The Role of Mg2+ Cofactor in the Guanine Nucleotide Exchange and GTP Hydrolysis Reactions of Rho Family GTP-binding Proteins. Journal of Biological Chemistry 275, 25299-25307.
    連結：
37. 6. Hariharan, N., Quijada, P., Mohsin, S., Joyo, A., Samse, K., Monsanto, M., De La Torre, A., Avitabile, D., Ormachea, L., McGregor, M.J., et al. (2015). Nucleostemin Rejuvenates Cardiac Progenitor Cells and Antagonizes Myocardial Aging. Journal of the American College of Cardiology 65, 133-147.
38. 19. Ohmura, M., Naka, K., Hoshii, T., Muraguchi, T., Shugo, H., Tamase, A., Uema, N., Ooshio, T., Arai, F., Takubo, K., et al. (2008). Identification of Stem Cells During Prepubertal Spermatogenesis via Monitoring of Nucleostemin Promoter Activity. STEM CELLS 26, 3237-3246.
39. 29. Shugo, H., Ooshio, T., Naito, M., Naka, K., Hoshii, T., Tadokoro, Y., Muraguchi, T., Tamase, A., Uema, N., Yamashita, T., et al. (2012). Nucleostemin in Injury-Induced Liver Regeneration. Stem Cells and Development 21, 3044-3054.
40. 40. Zhao, Q., Saro, D., Sachpatzidis, A., Singh, T.R., Schlingman, D., Zheng, X.-F., Mack, A., Tsai, M.-S., Mochrie, S., Regan, L., et al. (2014). The MHF complex senses branched DNA by binding a pair of crossover DNA duplexes. Nature Communications 5, 2987.
41. 41. Zhao, W., Vaithiyalingam, S., San Filippo, J., Maranon, David G., Jimenez-Sainz, J., Fontenay, Gerald V., Kwon, Y., Leung, Stanley G., Lu, L., Jensen, Ryan B., et al. (2015). Promotion of BRCA2-Dependent Homologous Recombination by DSS1 via RPA Targeting and DNA Mimicry. Molecular Cell 59, 176-187.