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研究生:林盈均
研究生(外文):Ying-Jyun Lin
論文名稱:探討酵母菌Ndt80蛋白對減數分裂期DNA複製的可能調控功能
論文名稱(外文):Studies on the potential role of Ndt80 in the control of DNA replication during yeast meiosis
指導教授:董桂書
口試委員:王廷方冀宏源
口試日期:2015-02-02
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:分子與細胞生物學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:中文
論文頁數:76
中文關鍵詞:酵母菌減數分裂染色體複製
外文關鍵詞:yeastmeiosisNdt80DNA replication
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在減數分裂中,一次DNA複製之後連續進行二次細胞核分裂。然而,在二次分裂期之間如何控制DNA不再進行複製目前並不是很清楚。在出芽酵母菌中,Ndt80是一個在減數分裂特定表現蛋白,它是啟動染色體分離以及孢子形成的一個必要轉錄因子。我們發現,若在營養生長細胞中異位表現Ndt80,則細胞出芽會受到抑制而且無法進行DNA複製,這顯示在減數分裂中Ndt80也可能具有抑制DNA複製的能力。我們認為Ndt80可能參與減數分裂二次分裂期間不再發生一次DNA複製的調控。
為了證明Ndt80是否在減數分裂中具有抑制DNA複製的能力,我們使細胞在減數分裂DNA複製期(pre-meiotic DNA replication)之前提早表現NDT80。在起始減數分裂時利用IME1啟動子將NDT80提早表現,細胞仍然可以進行DNA複製,但大多數的細胞會停滯在第一次減數分裂期。若將NDT80提早表現在減數分裂前的培養(pre-meiotic incubation)時,此時大部分細胞處於後複製複合體期(post-RC state),則部分細胞的DNA複製受到抑制。然而,若將NDT80表現在G1時期的細胞,則看不到DNA複製被抑制的現象。由這些結果可以得知,Ndt80在減數分裂中也許能透過防止前複製複合體(pre-RC)形成而抑制DNA複製,但是當pre-RC存在時卻無法抑制DNA複製。


Meiosis is characterized by a single round of DNA replication followed by two rounds of chromosome segregation. However, the mechanism to prevent DNA replication between the two rounds of division is not clear. In the budding yeast, the meiosis-specific transcription factor Ndt80 is pivotal to nuclear divisions and spore formation. Interestingly, ectopic expression of NDT80 in vegetative cells blocked budding and DNA replication. It is possible that Ndt80 also has the ability to inhibit DNA replication during meiosis. Therefore, we propose that Ndt80 might be involved in repressing another round of DNA replication between meiosis I and meiosis II.
To test whether Ndt80 has the ability to inhibit replication in meiosis, we tried to precociously express NDT80 before the pre-meiotic DNA replication. We found cells that precociously expressed of NDT80 through the IME1 promoter at the initiation of meiosis could execute pre-meiotic DNA synthesis but were arrested at meiosis I. When NDT80 was expressed during the pre-meiotic incubation, in which most cells were at the post-RC stage, pre-meiotic DNA replication was partially repressed. However, the repression effect was not observed when NDT80 was expressed at the time that most cells were at G1 phase. These results suggest that Ndt80 might repress DNA replication by preventing pre-RC formation, but it could not repress DNA replication at the existing pre-RCs.

CHAPTER 1. INTRODUCTION ................................1
Meiosis Overview .......................................1
Initiation of Meiosis ..................................2
Cell Cycle Control of DNA Replication ..................3
1. Initiation of DNA replication in mitotic cells ......3
2. Pre-meiotic S phase .................................4
3. The control of once and only once DNA replication per cell cycle .............................................5
NDT80 ..................................................6
Specific aims ..........................................8

CHAPTER 2. MATERIALS AND METHODS .......................9
Strains, Media, and Genetic Methods ....................9
Molecular Biology Methods .............................10
DNA Preparation and Transformation ....................10
Plasmids construction .................................10
Yeast strain construction .............................12
Time course of meiotic cells ..........................14
Immunofluorescence of tubulin .........................15
DNA content analysis ..................................16

CHAPTER 3. RESULTS ....................................17
I. Ectopic expression of NDT80 at the initiation of meiosis through IME1 and IME2 promoters................17
II. Induction of NDT80 before initiation of meiosis through a GAL promoter ................................21
III. The Analysis of NDT80-bc on controlling meiotic DNA replication ...........................................25

CHAPTER 4. DISCUSSION .................................28
I. The timing of NDT80 expression is pivotal for efficient meiosis .....................................28
II. Cell cycle arrest between meiosis I and meiosis II in IME1p-NDT80 cells .....................................29
III. The ability of Ndt80 to repress pre-meiotic DNA replication depends on the status of the origins ......29
IV. Ndt80 only partially repressed pre-meiotic DNA replication when expressed at pre-meiotic incubation ..30
V. The mechanisms of Ndt80 in repressing DNA replication .......................................................31
VI. There are multiple inhibitory mechanisms that control once DNA replication in meiosis .......................32


REFERENCES ............................................34
TABLES ................................................41
FIGURES ...............................................51
APPENDIX ..............................................74



Anderson, S. F., Steber, C. M., Esposito, R. E., and Coleman, J. E. (1995). UME6, a negative regulator of meiosis in Saccharomyces cerevisiae, contains a C-terminal Zn2Cys6 binuclear cluster that binds the URS1 DNA sequence in a zinc-dependent manner. Protein Sci, 4, 1832-1843.

Aparicio, O. M., Weinstein, D. M., and Bell, S. P. (1997). Components and dynamics of DNA replication complexes in S. cerevisiae: redistribution of MCM proteins and Cdc45p during S phase. Cell, 91, 59-69.

Araki, H. (2010). Cyclin-dependent kinase-dependent initiation of chromosomal DNA replication. Curr Opin Cell Biol, 22, 766-771.

Bell, S. P., and Dutta, A. (2002). DNA replication in eukaryotic cells. Annu Rev Biochem, 71, 333-374.

Bell, S. P., and Stillman, B. (1992). ATP-dependent recognition of eukaryotic origins of DNA replication by a multiprotein complex. Nature, 357, 128-134.

Benjamin, K. R., Zhang, C., Shokat, K. M., and Herskowitz, I. (2003). Control of landmark events in meiosis by the CDK Cdc28 and the meiosis-specific kinase Ime2. Genes Dev, 17, 1524-1539.

Birnboim, H. C., and Doly, J. (1979). Rapid Alkaline Extraction Procedure for Screening Recombinant Plasmid DNA. Nucleic Acids Research, 7, 1513-1523.

Blow, J. J., and Dutta, A. (2005). Preventing re-replication of chromosomal DNA. Nat Rev Mol Cell Biol, 6, 476-486.

Boeke, J. D., LaCroute, F., and Fink, G. R. (1984). A positive selection for mutants lacking orotidine-5''-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. Mol Gen Genet, 197, 345-346.

Buckingham, L. E., Wang, H. T., Elder, R. T., McCarroll, R. M., Slater, M. R., and Esposito, R. E. (1990). Nucleotide sequence and promoter analysis of SPO13, a meiosis-specific gene of Saccharomyces cerevisiae. Proc Natl Acad Sci U S A, 87, 9406-9410.

Budd, M. E., Wittrup, K. D., Bailey, J. E., and Campbell, J. L. (1989). DNA polymerase I is required for premeiotic DNA replication and sporulation but not for X-ray repair in Saccharomyces cerevisiae. Mol Cell Biol, 9, 365-376.

Cha, R. S., Weiner, B. M., Keeney, S., Dekker, J., and Kleckner, N. (2000). Progression of meiotic DNA replication is modulated by interchromosomal interaction proteins, negatively by Spo11p and positively by Rec8p. Genes Dev, 14, 493-503.

Chu, S., DeRisi, J., Eisen, M., Mulholland, J., Botstein, D., Brown, P. O., and Herskowitz, I. (1998). The transcriptional program of sporulation in budding yeast. Science, 282, 699-705.

Chu, S., and Herskowitz, I. (1998). Gametogenesis in yeast is regulated by a transcriptional cascade dependent on Ndt80. Mol Cell, 1, 685-696.

Clifford, D. M., Marinco, S. M., and Brush, G. S. (2004). The meiosis-specific protein kinase Ime2 directs phosphorylation of replication protein A. J Biol Chem, 279, 6163-6170.

Collins, I., and Newlon, C. S. (1994). Chromosomal DNA-Replication Initiates at the Same Origins in Meiosis and Mitosis. Mol Cell Biol, 14, 3524-3534.

Diffley, J. F. (2004). Regulation of early events in chromosome replication. Curr Biol, 14, R778-786.

Diffley, J. F., and Cocker, J. H. (1992). Protein-DNA interactions at a yeast replication origin. Nature, 357, 169-172.

Diffley, J. F., Cocker, J. H., Dowell, S. J., and Rowley, A. (1994). Two steps in the assembly of complexes at yeast replication origins in vivo. Cell, 78, 303-316.

Dirick, L., Goetsch, L., Ammerer, G., and Byers, B. (1998). Regulation of meiotic S phase by Ime2 and a Clb5,6-associated kinase in Saccharomyces cerevisiae. Science, 281, 1854-1857.

Drury, L. S., Perkins, G., and Diffley, J. F. (2000). The cyclin-dependent kinase Cdc28p regulates distinct modes of Cdc6p proteolysis during the budding yeast cell cycle. Curr Biol, 10, 231-240.

Esposito, R. E., and Klapholz, S. (1981). Meiosis and ascospore development. In The Molecular Biology of the Yeast Saccharomyces, J. N. Strather, ed. (New York: Cold Spring Harbor Press), pp. 211-287.

Foiani, M., Nadjar-Boger, E., Capone, R., Sagee, S., Hashimshoni, T., and Kassir, Y. (1996). A meiosis-specific protein kinase, Ime2, is required for the correct timing of DNA replication and for spore formation in yeast meiosis. Mol Gen Genet, 253, 278-288.

Granot, D., Margolskee, J. P., and Simchen, G. (1989). A long region upstream of the IME1 gene regulates meiosis in yeast. Mol Gen Genet, 218, 308-314.

Green, B. M., and Li, J. J. (2005). Loss of rereplication control in Saccharomyces cerevisiae results in extensive DNA damage. Mol Biol Cell, 16, 421-432.

Gurevich, V., and Kassir, Y. (2010). A Switch from a Gradient to a Threshold Mode in the Regulation of a Transcriptional Cascade Promotes Robust Execution of Meiosis in Budding Yeast. Plos One, 5.

Guttmann-Raviv, N., Boger-Nadjar, E., Edri, I., and Kassir, Y. (2001). Cdc28 and Ime2 possess redundant functions in promoting entry into premeiotic DNA replication in Saccharomyces cerevisiae. Genetics, 159, 1547-1558.

Hepworth, S. R., Friesen, H., and Segall, J. (1998). NDT80 and the meiotic recombination checkpoint regulate expression of middle sporulation-specific genes in Saccharomyces cerevisiae. Mol Cell Biol, 18, 5750-5761.

Holt, L. J., Hutti, J. E., Cantley, L. C., and Morgan, D. O. (2007). Evolution of Ime2 phosphorylation sites on Cdk1 substrates provides a mechanism to limit the effects of the phosphatase Cdc14 in meiosis. Mol Cell, 25, 689-702.

Hua, H., Namdar, M., Ganier, O., Gregan, J., Mechali, M., and Kearsey, S. E. (2013). Sequential steps in DNA replication are inhibited to ensure reduction of ploidy in meiosis. Mol Biol Cell, 24, 578-587.

Inai, T., Yukawa, M., and Tsuchiya, E. (2007). Interplay between chromatin and trans-acting factors on the IME2 promoter upon induction of the gene at the onset of meiosis. Mol Cell Biol, 27, 1254-1263.

Ito, H., Fukuda, Y., Murata, K., and Kimura, A. (1983). Transformation of intact yeast cells treated with alkali cations. J Bacteriol, 153, 163-168.

Kassir, Y., Granot, D., and Simchen, G. (1988). IME1, a positive regulator gene of meiosis in S. cerevisiae. Cell, 52, 853-862.

Kominami, K., Sakata, Y., Sakai, M., and Yamashita, I. (1993). Protein kinase activity associated with the IME2 gene product, a meiotic inducer in the yeast Saccharomyces cerevisiae. Biosci Biotechnol Biochem, 57, 1731-1735.

Mandel, S., Robzyk, K., and Kassir, Y. (1994). IME1 gene encodes a transcription factor which is required to induce meiosis in Saccharomyces cerevisiae. Dev Genet, 15, 139-147.

McCarroll, R. M., and Esposito, R. E. (1994). SPO13 negatively regulates the progression of mitotic and meiotic nuclear division in Saccharomyces cerevisiae. Genetics, 138, 47-60.

Mitchell, A. P. (1994). Control of meiotic gene expression in Saccharomyces cerevisiae. Microbiol Rev, 58, 56-70.

Mitchell, A. P., Driscoll, S. E., and Smith, H. E. (1990). Positive control of sporulation-specific genes by the IME1 and IME2 products in Saccharomyces cerevisiae. Mol Cell Biol, 10, 2104-2110.

Nguyen, V. Q., Co, C., Irie, K., and Li, J. J. (2000). Clb/Cdc28 kinases promote nuclear export of the replication initiator proteins Mcm2-7. Curr Biol, 10, 195-205.

Nguyen, V. Q., Co, C., and Li, J. J. (2001). Cyclin-dependent kinases prevent DNA re-replication through multiple mechanisms. Nature, 411, 1068-1073.

Ofir, Y., Sagee, S., Guttmann-Raviv, N., Pnueli, L., and Kassir, Y. (2004). The role and regulation of the preRC component Cdc6 in the initiation of premeiotic DNA replication. Mol Biol Cell, 15, 2230-2242.

Pak, J., and Segall, J. (2002). Regulation of the premiddle and middle phases of expression of the NDT80 gene during sporulation of Saccharomyces cerevisiae. Mol Cell Biol, 22, 6417-6429.

Raithatha, S. A., and Stuart, D. T. (2009). A comparison of fluorescent DNA binding dyes for flow cytometric analysis of sporulating Saccharomyces cerevisiae. J Microbiol Methods, 78, 357-359.

Rubin-Bejerano, I., Mandel, S., Robzyk, K., and Kassir, Y. (1996). Induction of meiosis in Saccharomyces cerevisiae depends on conversion of the transcriptional represssor Ume6 to a positive regulator by its regulated association with the transcriptional activator Ime1. Mol Cell Biol, 16, 2518-2526.

Sagee, S., Sherman, A., Shenhar, G., Robzyk, K., Ben-Doy, N., Simchen, G., and Kassir, Y. (1998). Multiple and distinct activation and repression sequences mediate the regulated transcription of IME1, a transcriptional activator of meiosis-specific genes in Saccharomyces cerevisiae. Mol Cell Biol, 18, 1985-1995.

Shin, M. E., Skokotas, A., and Winter, E. (2010). The Cdk1 and Ime2 protein kinases trigger exit from meiotic prophase in Saccharomyces cerevisiae by inhibiting the Sum1 transcriptional repressor. Mol Cell Biol, 30, 2996-3003.

Simchen, G. (1974). Are mitotic functions required in meiosis? Genetics, 76, 745-753.

Smith, H. E., Driscoll, S. E., Sia, R. A., Yuan, H. E., and Mitchell, A. P. (1993). Genetic evidence for transcriptional activation by the yeast IME1 gene product. Genetics, 133, 775-784.

Smith, H. E., and Mitchell, A. P. (1989). A transcriptional cascade governs entry into meiosis in Saccharomyces cerevisiae. Mol Cell Biol, 9, 2142-2152.

Sopko, R., Raithatha, S., and Stuart, D. (2002). Phosphorylation and maximal activity of Saccharomyces cerevisiae meiosis-specific transcription factor Ndt80 is dependent on Ime2. Mol Cell Biol, 22, 7024-7040.

Stillman, B. (1996). Cell cycle control of DNA replication. Science, 274, 1659-1664.

Strich, R., Surosky, R. T., Steber, C., Dubois, E., Messenguy, F., and Esposito, R. E. (1994). UME6 is a key regulator of nitrogen repression and meiotic development. Genes Dev, 8, 796-810.

Szwarcwort-Cohen, M., Gurevich, V., Sagee, S., and Kassir, Y. (2010). Ectopic expression of human Cdk2 and its yeast homolog, Ime2, is deleterious to Saccharomyces cerevisiae. Cell Cycle, 9, 4711-4719.

Tanaka, S., and Araki, H. (2010). Regulation of the initiation step of DNA replication by cyclin-dependent kinases. Chromosoma, 119, 565-574.

Teng, F.-H. (2005). Mechanism for repressing DNA replication by Ndt80. Master Thesis. Nactional Taiwan University. Taiwan.

Toone, W. M., Aerne, B. L., Morgan, B. A., and Johnston, L. H. (1997). Getting started: regulating the initiation of DNA replication in yeast. Annu Rev Microbiol, 51, 125-149.

Tung, K. S., Hong, E. J., and Roeder, G. S. (2000). The pachytene checkpoint prevents accumulation and phosphorylation of the meiosis-specific transcription factor Ndt80. Proc Natl Acad Sci U S A, 97, 12187-12192.

Wang, Y., Chang, C. Y., Wu, J. F., and Tung, K. S. (2011). Nuclear localization of the meiosis-specific transcription factor Ndt80 is regulated by the pachytene checkpoint. Mol Biol Cell, 22, 1878-1886.

Wilmes, G. M., Archambault, V., Austin, R. J., Jacobson, M. D., Bell, S. P., and Cross, F. R. (2004). Interaction of the S-phase cyclin Clb5 with an "RXL" docking sequence in the initiator protein Orc6 provides an origin-localized replication control switch. Genes Dev, 18, 981-991.

Xie, J. X., Pierce, M., Gailus-Durner, V., Wagner, M., Winter, E., and Vershon, A. K. (1999). Sum1 and Hst1 repress middle sporulation-specific gene expression during mitosis in Saccharomyces cerevisiae. Embo Journal, 18, 6448-6454.

Xu, L., Ajimura, M., Padmore, R., Klein, C., and Kleckner, N. (1995). NDT80, a meiosis-specific gene required for exit from pachytene in Saccharomyces cerevisiae. Mol Cell Biol, 15, 6572-6581.

Yanow, S. K., Lygerou, Z., and Nurse, P. (2001). Expression of Cdc18/Cdc6 and Cdt1 during G2 phase induces initiation of DNA replication. EMBO J, 20, 4648-4656.

Yekezare, M., Gomez-Gonzalez, B., and Diffley, J. F. (2013). Controlling DNA replication origins in response to DNA damage - inhibit globally, activate locally. J Cell Sci, 126, 1297-1306.

Yoshida, M., Kawaguchi, H., Sakata, Y., Kominami, K., Hirano, M., Shima, H., Akada, R., and Yamashita, I. (1990). Initiation of meiosis and sporulation in Saccharomyces cerevisiae requires a novel protein kinase homologue. Mol Gen Genet, 221, 176-186.

Zamb, T. J., and Roth, R. (1977). Role of Mitotic Replication Genes in Chromosome Duplication during Meiosis - (Yeast-DNA Initiation DNA Polymerization). Proc Natl Acad Sci U S A, 74, 3951-3955.



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