請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/70374
完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 冀宏源(Hung-Yuan Chi) | |
dc.contributor.author | Hao-Yen Chang | en |
dc.contributor.author | 張皓衍 | zh_TW |
dc.date.accessioned | 2021-06-17T04:26:50Z | - |
dc.date.available | 2019-08-18 | |
dc.date.copyright | 2018-08-18 | |
dc.date.issued | 2018 | |
dc.date.submitted | 2018-08-14 | |
dc.identifier.citation | 1. Bell, J.C. and S.C. Kowalczykowski, Mechanics and Single-Molecule Interrogation of DNA Recombination. Annu Rev Biochem, 2016. 85: p. 193-226.
2. Kowalczykowski, S.C., An Overview of the Molecular Mechanisms of Recombinational DNA Repair. Cold Spring Harb Perspect Biol, 2015. 7(11). 3. Zhao, W., et al., Promotion of BRCA2-Dependent Homologous Recombination by DSS1 via RPA Targeting and DNA Mimicry. Mol Cell, 2015. 59(2): p. 176-87. 4. Sung, P. and H. Klein, Mechanism of homologous recombination: mediators and helicases take on regulatory functions. Nat Rev Mol Cell Biol, 2006. 7(10): p. 739-50. 5. Bizard, A.H. and I.D. Hickson, The dissolution of double Holliday junctions. Cold Spring Harb Perspect Biol, 2014. 6(7): p. a016477. 6. Cloud, V., et al., Rad51 is an accessory factor for Dmc1-mediated joint molecule formation during meiosis. Science, 2012. 337(6099): p. 1222-5. 7. Bell, J.C. and S.C. Kowalczykowski, RecA: Regulation and Mechanism of a Molecular Search Engine. Trends Biochem Sci, 2016. 41(6): p. 491-507. 8. Yu, X., et al., Domain structure and dynamics in the helical filaments formed by RecA and Rad51 on DNA. Proc Natl Acad Sci U S A, 2001. 98(15): p. 8419-24. 9. Sung, P. and D.L. Robberson, DNA strand exchange mediated by a RAD51-ssDNA nucleoprotein filament with polarity opposite to that of RecA. Cell, 1995. 82(3): p. 453-61. 10. Ogawa, T., et al., Similarity of the yeast RAD51 filament to the bacterial RecA filament. Science, 1993. 259(5103): p. 1896-9. 11. Cox, M.M., The bacterial RecA protein as a motor protein. Annu Rev Microbiol, 2003. 57: p. 551-77. 12. Bianco, P.R., R.B. Tracy, and S.C. Kowalczykowski, DNA strand exchange proteins: a biochemical and physical comparison. Front Biosci, 1998. 3: p. D570-603. 13. Robertson, R.B., et al., Structural transitions within human Rad51 nucleoprotein filaments. Proc Natl Acad Sci U S A, 2009. 106(31): p. 12688-93. 14. Hilario, J., et al., Direct imaging of human Rad51 nucleoprotein dynamics on individual DNA molecules. Proc Natl Acad Sci U S A, 2009. 106(2): p. 361-8. 15. Chi, P., et al., Roles of ATP binding and ATP hydrolysis in human Rad51 recombinase function. DNA Repair (Amst), 2006. 5(3): p. 381-91. 16. Bugreev, D.V. and A.V. Mazin, Ca2+ activates human homologous recombination protein Rad51 by modulating its ATPase activity. Proc Natl Acad Sci U S A, 2004. 101(27): p. 9988-93. 17. Krogh, B.O. and L.S. Symington, Recombination proteins in yeast. Annu Rev Genet, 2004. 38: p. 233-71. 18. San Filippo, J., P. Sung, and H. Klein, Mechanism of eukaryotic homologous recombination. Annu Rev Biochem, 2008. 77: p. 229-57. 19. Heyer, W.D., K.T. Ehmsen, and J. Liu, Regulation of homologous recombination in eukaryotes. Annu Rev Genet, 2010. 44: p. 113-39. 20. Moynahan, M.E. and M. Jasin, Mitotic homologous recombination maintains genomic stability and suppresses tumorigenesis. Nat Rev Mol Cell Biol, 2010. 11(3): p. 196-207. 21. Aguilera, A. and T. Garcia-Muse, Causes of genome instability. Annu Rev Genet, 2013. 47: p. 1-32. 22. Malkova, A. and J.E. Haber, Mutations arising during repair of chromosome breaks. Annu Rev Genet, 2012. 46: p. 455-73. 23. Cole, F., S. Keeney, and M. Jasin, Preaching about the converted: how meiotic gene conversion influences genomic diversity. Ann N Y Acad Sci, 2012. 1267: p. 95-102. 24. Neale, M.J. and S. Keeney, Clarifying the mechanics of DNA strand exchange in meiotic recombination. Nature, 2006. 442(7099): p. 153-8. 25. Sehorn, M.G. and P. Sung, Meiotic recombination: an affair of two recombinases. Cell Cycle, 2004. 3(11): p. 1375-7. 26. Hong, S., et al., The logic and mechanism of homologous recombination partner choice. Mol Cell, 2013. 51(4): p. 440-53. 27. Lao, J.P., et al., Meiotic crossover control by concerted action of Rad51-Dmc1 in homolog template bias and robust homeostatic regulation. PLoS Genet, 2013. 9(12): p. e1003978. 28. Hayase, A., et al., A protein complex containing Mei5 and Sae3 promotes the assembly of the meiosis-specific RecA homolog Dmc1. Cell, 2004. 119(7): p. 927-40. 29. Tsuzuki, T., et al., Targeted disruption of the Rad51 gene leads to lethality in embryonic mice. Proc Natl Acad Sci U S A, 1996. 93(13): p. 6236-40. 30. Yoshida, K., et al., The mouse RecA-like gene Dmc1 is required for homologous chromosome synapsis during meiosis. Mol Cell, 1998. 1(5): p. 707-18. 31. Pittman, D.L., et al., Meiotic prophase arrest with failure of chromosome synapsis in mice deficient for Dmc1, a germline-specific RecA homolog. Mol Cell, 1998. 1(5): p. 697-705. 32. Lim, D.S. and P. Hasty, A mutation in mouse rad51 results in an early embryonic lethal that is suppressed by a mutation in p53. Mol Cell Biol, 1996. 16(12): p. 7133-43. 33. Sheridan, S.D., et al., A comparative analysis of Dmc1 and Rad51 nucleoprotein filaments. Nucleic Acids Res, 2008. 36(12): p. 4057-66. 34. Yu, X. and E.H. Egelman, Helical filaments of human Dmc1 protein on single-stranded DNA: a cautionary tale. J Mol Biol, 2010. 401(3): p. 544-51. 35. Li, Z., et al., Recombination activities of HsDmc1 protein, the meiotic human homolog of RecA protein. Proc Natl Acad Sci U S A 1997. 94(21): p. 11221-6. 36. Gupta, R.C., et al., The synaptic activity of HsDmc1, a human recombination protein specific to meiosis. Proc Natl Acad Sci U S A, 2001. 98(15): p. 8433-9. 37. Sehorn, M.G., et al., Human meiotic recombinase Dmc1 promotes ATP-dependent homologous DNA strand exchange. Nature, 2004. 429(6990): p. 433-7. 38. Baumann, P., F.E. Benson, and S.C. West, Human Rad51 protein promotes ATP-dependent homologous pairing and strand transfer reactions in vitro. Cell, 1996. 87(4): p. 757-66. 39. Fung, C.W., et al., The rad51-K191R ATPase-defective mutant is impaired for presynaptic filament formation. Mol Cell Biol, 2006. 26(24): p. 9544-54. 40. Li, X., et al., Rad51 and Rad54 ATPase activities are both required to modulate Rad51-dsDNA filament dynamics. Nucleic Acids Res, 2007. 35(12): p. 4124-40. 41. Sigurdsson, S., et al., Homologous DNA pairing by human recombination factors Rad51 and Rad54. J Biol Chem, 2002. 277(45): p. 42790-4. 42. Rossi, M.J., et al., The RecA/RAD51 protein drives migration of Holliday junctions via polymerization on DNA. Proc Natl Acad Sci U S A, 2011. 108(16): p. 6432-7. 43. Amunugama, R., et al., RAD51 protein ATP cap regulates nucleoprotein filament stability. J Biol Chem, 2012. 287(12): p. 8724-36. 44. Tsai, S.P., et al., Rad51 presynaptic filament stabilization function of the mouse Swi5-Sfr1 heterodimeric complex. Nucleic Acids Res, 2012. 40(14): p. 6558-69. 45. Su, G.C., et al., Enhancement of ADP release from the RAD51 presynaptic filament by the SWI5-SFR1 complex. Nucleic Acids Res, 2014. 42(1): p. 349-58. 46. Bugreev, D.V., et al., Activation of human meiosis-specific recombinase Dmc1 by Ca2+. J Biol Chem, 2005. 280(29): p. 26886-95. 47. Sharma, D., et al., Role of the conserved lysine within the Walker A motif of human DMC1. DNA Repair (Amst), 2013. 12(1): p. 53-62. 48. Tombline, G., K.S. Shim, and R. Fishel, Biochemical characterization of the human RAD51 protein. II. Adenosine nucleotide binding and competition. J Biol Chem, 2002. 277(17): p. 14426-33. 49. Rai, V., et al., Conserved Asp327 of walker B motif in the N-terminal nucleotide binding domain (NBD-1) of Cdr1p of Candida albicans has acquired a new role in ATP hydrolysis. Biochemistry, 2006. 45(49): p. 14726-39. 50. Stratford, F.L., et al., The Walker B motif of the second nucleotide-binding domain (NBD2) of CFTR plays a key role in ATPase activity by the NBD1-NBD2 heterodimer. Biochem J, 2007. 401(2): p. 581-6. 51. Chiraniya, A., et al., A novel function for the conserved glutamate residue in the walker B motif of replication factor C. Genes (Basel), 2013. 4(2): p. 134-51. 52. Zhao, W., et al., Mechanistic insights into the role of Hop2-Mnd1 in meiotic homologous DNA pairing. Nucleic Acids Res, 2014. 42(2): p. 906-17. 53. Pezza, R.J., et al., Hop2/Mnd1 acts on two critical steps in Dmc1-promoted homologous pairing. Genes Dev, 2007. 21(14): p. 1758-66. 54. Bugreev, D.V., et al., HOP2-MND1 modulates RAD51 binding to nucleotides and DNA. Nat Commun, 2014. 5: p. 4198. 55. Jackson, A.P., et al., Identification of microcephalin, a protein implicated in determining the size of the human brain. Am J Hum Genet, 2002. 71(1): p. 136-42. 56. Duerinckx, S. and M. Abramowicz, The genetics of congenitally small brains. Semin Cell Dev Biol, 2018. 76: p. 76-85. 57. Lin, S.-Y. and S.J. Elledge, Multiple Tumor Suppressor Pathways Negatively Regulate Telomerase. Cell, 2003. 113(7): p. 881-889. 58. Venkatesh, T. and P.S. Suresh, Emerging roles of MCPH1: expedition from primary microcephaly to cancer. Eur J Cell Biol, 2014. 93(3): p. 98-105. 59. Rai, R., et al., BRIT1 regulates early DNA damage response, chromosomal integrity, and cancer. Cancer Cell, 2006. 10(2): p. 145-57. 60. Richardson, J., et al., Microcephalin is a new novel prognostic indicator in breast cancer associated with BRCA1 inactivation. Breast Cancer Res Treat, 2011. 127(3): p. 639-48. 61. Jo, Y.H., et al., MCPH1 protein expression and polymorphisms are associated with risk of breast cancer. Gene, 2013. 517(2): p. 184-90. 62. Bhattacharya, N., et al., Frequent alterations of MCPH1 and ATM are associated with primary breast carcinoma: clinical and prognostic implications. Ann Surg Oncol, 2013. 20 Suppl 3: p. S424-32. 63. Lin, S.Y., Y. Liang, and K. Li, Multiple roles of BRIT1/MCPH1 in DNA damage response, DNA repair, and cancer suppression. Yonsei Med J, 2010. 51(3): p. 295-301. 64. Gavvovidis, I., et al., A novel MCPH1 isoform complements the defective chromosome condensation of human MCPH1-deficient cells. PLoS One, 2012. 7(8): p. e40387. 65. Kumar, A., M. Markandaya, and S.C. Girimaji, Primary microcephaly: microcephalin and ASPM determine the size of the human brain. J Biosci, 2002. 27(7): p. 629-32. 66. O'Driscoll, M., A.P. Jackson, and P.A. Jeggo, Microcephalin: a causal link between impaired damage response signalling and microcephaly. Cell Cycle, 2006. 5(20): p. 2339-44. 67. Wood, J.L., et al., MCPH1 functions in an H2AX-dependent but MDC1-independent pathway in response to DNA damage. J Biol Chem, 2007. 282(48): p. 35416-23. 68. Wood, J.L., et al., Microcephalin/MCPH1 associates with the Condensin II complex to function in homologous recombination repair. J Biol Chem, 2008. 283(43): p. 29586-92. 69. Wu, X., et al., Microcephalin regulates BRCA2 and Rad51-associated DNA double-strand break repair. Cancer Res, 2009. 69(13): p. 5531-6. 70. Gavvovidis, I., et al., MCPH1 patient cells exhibit delayed release from DNA damage-induced G2/M checkpoint arrest Cell Cycle, 2010. 9(24): p. 4893-4899. 71. Zhang, B., et al., Phosphorylation of the BRCA1 C terminus (BRCT) repeat inhibitor of hTERT (BRIT1) protein coordinates TopBP1 protein recruitment and amplifies ataxia telangiectasia-mutated and Rad3-related (ATR) Signaling. J Biol Chem, 2014. 289(49): p. 34284-95. 72. Liang, Y., et al., BRIT1/MCPH1 is essential for mitotic and meiotic recombination DNA repair and maintaining genomic stability in mice. PLoS Genet, 2010. 6(1): p. e1000826. 73. Gruber, R., et al., MCPH1 regulates the neuroprogenitor division mode by coupling the centrosomal cycle with mitotic entry through the Chk1-Cdc25 pathway. Nat Cell Biol, 2011. 13(11): p. 1325-34. 74. Zhou, Z.W., et al., DNA damage response in microcephaly development of MCPH1 mouse model. DNA Repair (Amst), 2013. 12(8): p. 645-55. 75. Liang, Y., et al., Mcph1/Brit1 deficiency promotes genomic instability and tumor formation in a mouse model. Oncogene, 2015. 34(33): p. 4368-78. 76. Alderton, G.K., et al., Regulation of mitotic entry by microcephalin and its overlap with ATR signalling. Nat Cell Biol, 2006. 8(7): p. 725-33. 77. Shao, Z., et al., Specific recognition of phosphorylated tail of H2AX by the tandem BRCT domains of MCPH1 revealed by complex structure. J Struct Biol, 2012. 177(2): p. 459-68. 78. Singh, N., et al., Dual recognition of phosphoserine and phosphotyrosine in histone variant H2A.X by DNA damage response protein MCPH1. Proc Natl Acad Sci U S A, 2012. 109(36): p. 14381-6. 79. Jeffers, L.J., et al., Distinct BRCT domains in Mcph1/Brit1 mediate ionizing radiation-induced focus formation and centrosomal localization. Oncogene, 2008. 27(1): p. 139-44. 80. Ge, C., L. Che, and C. Du, The UBC Domain Is Required for BRUCE to Promote BRIT1/MCPH1 Function in DSB Signaling and Repair Post Formation of BRUCE-USP8-BRIT1 Complex. PLoS One, 2015. 10(12): p. e0144957. 81. Ge, C., et al., BRUCE regulates DNA double-strand break response by promoting USP8 deubiquitination of BRIT1. Proc Natl Acad Sci U S A, 2015. 112(11): p. E1210-9. 82. Peng, G., et al., BRIT1/MCPH1 links chromatin remodelling to DNA damage response. Nat Cell Biol, 2009. 11(7): p. 865-72. 83. Chang, H.Y., et al., Functional Relationship of ATP Hydrolysis, Presynaptic Filament Stability, and Homologous DNA Pairing Activity of the Human Meiotic Recombinase DMC1. J Biol Chem, 2015. 290(32): p. 19863-73. 84. Lu, C.H., et al., Stable Nuclei of Nucleoprotein Filament and High ssDNA Binding Affinity Contribute to Enhanced RecA E38K Recombinase Activity. Sci Rep, 2017. 7(1): p. 14964. 85. Shi, L., M. Li, and B. Su, MCPH1/BRIT1 represses transcription of the human telomerase reverse transcriptase gene. Gene, 2012. 495(1): p. 1-9. 86. Liu, Y., et al., Conformational changes modulate the activity of human RAD51 protein. J Mol Biol, 2004. 337(4): p. 817-27. 87. Lin, S.Y., et al., BRIT1/MCPH1 is a DNA damage responsive protein that regulates the Brca1-Chk1 pathway, implicating checkpoint dysfunction in microcephaly. Proc Natl Acad Sci U S A, 2005. 102(42): p. 15105-9. 88. Pan, M.R., et al., Chromodomain helicase DNA-binding protein 4 (CHD4) regulates homologous recombination DNA repair, and its deficiency sensitizes cells to poly(ADP-ribose) polymerase (PARP) inhibitor treatment. J Biol Chem, 2012. 287(9): p. 6764-72. 89. Hirano, T., Condensins: universal organizers of chromosomes with diverse functions. Genes Dev, 2012. 26(15): p. 1659-78. 90. Nakanishi, A., et al., Interference with BRCA2, which localizes to the centrosome during S and early M phase, leads to abnormal nuclear division. Biochem Biophys Res Commun, 2007. 355(1): p. 34-40. 91. Niwa, T., et al., BRCA2 interacts with the cytoskeletal linker protein plectin to form a complex controlling centrosome localization. Cancer Sci, 2009. 100(11): p. 2115-25. 92. Wang, H.F., et al., BRCA2 and nucleophosmin coregulate centrosome amplification and form a complex with the Rho effector kinase ROCK2. Cancer Res, 2011. 71(1): p. 68-77. 93. Trimborn, M., et al., Establishment of a mouse model with misregulated chromosome condensation due to defective Mcph1 function. PLoS One, 2010. 5(2): p. e9242. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/70374 | - |
dc.description.abstract | 當同源重組酵素RAD51透過同源重組修復DNA 雙股斷裂時,透過調控RAD51的ATP水解使得RAD51保持在ATP結合的活化態以穩定RAD51的核蛋白絲,對後續的雙股交換具有增強的作用。然而,對於減數分裂時期的同源重組酵素DMC1而言,調控ATP水解乃至於DMC1的核蛋白絲穩定,以及後續的雙股交換之間的關聯性仍是未知。為了探討這三者的關聯性,我們建立了蛋白表現及純化的步驟,得到人類DMC1正常蛋白與D317K點突變蛋白。經由我們的生化研究指出,在保有ATP結合的能力下,ATP水解抑制會促進DMC1核蛋白絲的穩定;然而,我們發現DMC1的雙股交換能力並未隨之提升,對於DMC1而言,單純ATP水解抑制並不能得到活化的DMC1核蛋白絲,顯示了減數分裂的同源重組酵素DMC1與同源重組酵素RAD51有相當大的不同。除了調控水解之外,我們也有興趣從疾病關聯性來尋找影響RAD51的核蛋白絲的調控因子。小頭症蛋白被發現與RAD51在細胞內有效結合DNA受損部位有關;因此,我們假設小頭症蛋白透過穩定RAD51的核蛋白絲來協助基因修補。為了驗證假設,我們首次建立了小頭症蛋白表現及純化的方法。透過生化研究顯示,我們意外發現小頭症蛋白具有DNA結合的能力,並且能直接結合RAD51;更重要的,透過不同研究方法,我們證明小頭症蛋白能直接穩定RAD51的核蛋白絲。 | zh_TW |
dc.description.abstract | During repairing DNA double-strand breaks by RAD51-mediated homologous recombination, the presynaptic filament is maintained by regulating ATP hydrolysis of RAD51 to stay in ATP-bound form, which enhances the subsequent strand exchange. However, for meiotic specific homologous recombinase DMC1, the association between regulation of ATP hydrolysis and even nucleoprotein stabilization of DMC1, and subsequent DNA exchange is still unknown. To explore this issue, we established the protein expression and purification to obtain human DMC1 wild-type and D317K point mutant proteins. Through our biochemical studies, ATP hydrolysis inhibition promotes the stability of DMC1 presynaptic filaments while retaining ATP binding capacity; however, we found that DNA strand exchange capacity of DMC1 did not increase. For DMC1, simply ATP hydrolysis inhibition did not result in the activation of DMC1 nucleoprotein filaments, indicating that the meiotic homologous recombinase DMC1 is quite different from the homologous recombinase RAD51. Except for regulating ATP hydrolysis, we are also interested in finding the accessory factors regarding stabilizing RAD51 presynaptic filaments from disease association. Microcephalin 1 (MCPH1) had been found to be involved in the efficient binding of RAD51 to damaged sites in cells; therefore, we hypothesized that MCPH1 stabilizing RAD51 presynaptic filaments. To validate the hypothesis, we first established a method for expression and purification of MCPH1 protein. Through biochemical studies, surprisingly, we have discovered that MCPH1 protein has the ability to bind DNA and can directly bind to RAD51. Importantly, through different approaches, we have demonstrated that MCPH1 can stabilize presynaptic filaments of RAD51. | en |
dc.description.provenance | Made available in DSpace on 2021-06-17T04:26:50Z (GMT). No. of bitstreams: 1 ntu-107-D99b46013-1.pdf: 4138919 bytes, checksum: dc064d42c9b8e91992f7e5d378dcd03b (MD5) Previous issue date: 2018 | en |
dc.description.tableofcontents | 中文摘要 1
ABSTRACT 2 LITERATURE REVIEW AND GENERAL INTRODUCTION 7 Chapter 1. Functional Relationship of ATP Hydrolysis, Presynaptic Filament Stability, and Homologous DNA Pairing Activity of the Human Meiotic Recombinase DMC1 10 1-1. ABSTRACT 10 1-2. INTRODUCTION 12 1-2-1. The biological functions of homologous recombination 12 1-2-2. Cooperation of RAD51 and DMC1 in meiosis 12 1-2-3. The known properties of ATP hydrolysis of RAD51 and DMC1-mediated homologous recombination in vitro 13 1-2-4. The active conformation of DMC1 was not correlated with ATP hydrolysis 14 1-3. MATERIAL AND METHODS 16 1-3-1. DNA substrates 16 1-3-2. Plasmids 17 1-3-3. Protein expression and purification 17 1-3-4. UV cross-linking assay 19 1-3-5. ATPase assay 19 1-3-6. DNA mobility shift assay 20 1-3-7. Exonuclease protection assay 21 1-3-8. DNA strand exchange assay 22 1-3-9. D-loop assay 22 1-4. RESULTS 24 1-4-1. Construction of DMC1 mutants 24 1-4-2. The DMC1 D317K mutant is attenuated for ATP hydrolysis 25 1-4-3. DNA-binding properties of the DMC1 D317K mutant 26 1-4-4. Relationship between ATP hydrolysis and presynaptic filament stability 27 1-4-5. Dependence of DMC1-mediated homologous DNA pairing on ATP hydrolysis 28 1-5. DISCUSSION 30 1-5-1. Dependence of the DMC1 recombinase activity on ATP binding and hydrolysis 30 1-5-2. Differences in functional attributes between RAD51 and DMC1 31 1-6. FIGURES 34 Chapter 2. Studying the functional interaction of MCPH1 and RAD51 44 2-1. ABSTRACT 44 2-2. INTRODUCTION 46 2-2-1. MCPH1 in microcephaly and cancer 46 2-2-2. Expression, structure, and distribution 47 2-2-3. MCPH1 in mouse model organism 48 2-2-4. MCPH1 in DNA damage response 49 2-2-5. MCPH1 in homologous recombination 50 2-2-6. Aim of the study 51 2-3. MATERIALS AND METHODS 53 2-3-1. DNA substrates 53 2-3-2. Plasmids 53 2-3-3. Protein expression 54 2-3-4. Protein purification 55 2-3-5. Gel-filtration analysis 57 2-3-6. DNA mobility shift assay 57 2-3-7. Affinity pulldown 58 2-3-8. Benzonase protection assay 59 2-3-9. Tethering Particle Motion 60 2-4. RESULTS 61 2-4-1. Induction, purification, and identification of mouse MCPH1 61 2-4-2. MCPH1 is a bona fide DNA-binding protein 62 2-4-3. MCPH1 physically interacts with RAD51 or DMC1 63 2-4-4. MCPH1 stabilizes RAD51-ssDNA filament 64 2-4-5. MCPH1 prevents RAD51 filament disassembly 65 2-4-6. Multiple contacts between MCPH1 and RAD51 66 2-5. DISCUSSION 67 2-5-1. Establish in vitro system to study functional interaction between MCPH1 and RAD51 67 2-5-2. Implication of MCPH1 DNA-binding property 67 2-5-3. Stabilization of RAD51 presynaptic filament 68 2-5-4. Cooperation with BRCA2 69 2-6. FIGURES 71 APPENDIX 79 REFERENCES 82 ------------------------------------------------------------- LIST OF FIGURES Chapter 1. Functional Relationship of ATP Hydrolysis, Presynaptic Filament Stability, and Homologous DNA Pairing Activity of the Human Meiotic Recombinase DMC1 1-6. FIGURES 34 FIGURE 1. Purification of human DMC1 and mutant proteins 34 FIGURE 2. ATP binding and hydrolysis by DMC1 and mutant proteins 35 FIGURE 3. DNA-binding activity of DMC1 and DMC1 D317K 37 FIGURE 4. Determination of DMC1 presynaptic filament stability by protection against Exonuclease I 38 FIGURE 5. Determination of DMC1 presynaptic filament stability by protection against RecJ 39 FIGURE 6. DNA strand exchange in the absence of ATP hydrolysis 40 FIGURE 7. D-loop formation in the absence of ATP hydrolysis 41 FIGURE 8. Defects of the DMC1 K132R and D223N mutants in presynaptic filament formation and DNA strand exchange 42 TABLE 1. Comparison of ATPase activity, filament stability and recombinase activity of DMC1 and RAD51 43 Chapter 2. Studying the functional interaction of MCPH1 and RAD51 2-6. FIGURES 71 FIGURE 9. Expression and purification of mouse MCPH1 protein 71 FIGURE 10. Identity of purified MCPH1 through mass spectrometry 72 FIGURE 11. MCPH1 forms a soluble heterogeneous oligomer in solution 73 FIGURE 12. DNA binding property of MCPH1 74 FIGURE 13. MCPH1 physical interacts with RAD51 and DMC1 75 FIGURE 14. MCPH1 stabilizes RAD51 presynaptic filament 76 FIGURE 15. MCPH1 prevents RAD51-ssDNA filament disassembly 77 FIGURE 16. Interaction of MCPH1 with RAD51 is multiple contacts 78 APPENDIX FIGURE A. Molecular mechanism of homologous recombination 79 FIGURE B. The function of BRCA2 in the formation of RAD51 filament 80 FIGURE C. Recombinase filament and displacement-loop formation 81 | |
dc.language.iso | en | |
dc.title | 研究重組酵素結合去氧核糖核酸之調控 | zh_TW |
dc.title | Studying the regulations of recombinases on DNA filament assembly | en |
dc.type | Thesis | |
dc.date.schoolyear | 106-2 | |
dc.description.degree | 博士 | |
dc.contributor.oralexamcommittee | 唐堂(Tang K Tang),廖泓鈞(Hungjiun Liaw),王中茹(Chung-Ju Wang),潘美仁(Mei-Ren Pan),吳青錫(Ching-Shyi Wu) | |
dc.subject.keyword | 同源重組,DMC1,RAD51,ATP 水解,小頭症蛋白 MCPH1, | zh_TW |
dc.subject.keyword | Homologous recombination,DMC1,RAD51,ATP hydrolysis,Microcephalin 1, | en |
dc.relation.page | 88 | |
dc.identifier.doi | 10.6342/NTU201803267 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2018-08-14 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 生化科學研究所 | zh_TW |
顯示於系所單位: | 生化科學研究所 |
文件中的檔案:
檔案 | 大小 | 格式 | |
---|---|---|---|
ntu-107-1.pdf 目前未授權公開取用 | 4.04 MB | Adobe PDF |
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。