Zagoriy I. The role of polo-like kinase in regulating. The prophase-to-metaphase transition of meiosis I: Diss. … Dr. rer. nat. ([Dresden], [2013]). - ОГЛАВЛЕНИЕ / CONTENTS

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ОбложкаZagoriy I. The role of polo-like kinase in regulating. The prophase-to-metaphase transition of meiosis I: Diss. … Dr. rer. nat. - [Dresden]: [Technische Universitat Dresden, 2013]. - 82 l.: ill. - Bibliogr.: l.69-81.
 

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Оглавление / Contents
 
Abstract
1    Introduction ............................................... 1
1.1  Overview of the genome stability maintenance mechanisms .... 1
     1.1.1  Sister chromatid cohesion ........................... 2
     1.1.2  Spindle assembly checkpoint and chromosome
            segregation ......................................... 3
     1.1.3  DNA damage checkpoint and double strand break
            repair .............................................. 4
1.2  Maintenance of genome stability in meiosis ................. 6
     1.2.1  Pre-meiotic DNA replication and cohesin loading ..... 6
     1.2.2  Meiotic inter-homolog recombination and crossover
            formation ........................................... 7
     1.2.3  Meiotic recombination checkpoint .................... 9
            1.2.3.1  Transduction of the checkpoint signals .... 10
            1.2.3.2  Mechanism of the meiotic recombination
                     checkpoint-mediated arrest ................ 11
     1.2.4  Synaptonemal complex assembly and architecture ..... 11
            1.2.4.1  Components of the synaptonemal complex
                     of S. cerevisiae .......................... 11
            1.2.4.2  Synapsis initiation and crossover
                     interference .............................. 12
     1.2.5  Linkage of homologous chromosomes via chiasmata .... 13
     1.2.6  Mono-orientation of sister kinetochores ............ 14
     1.2.7  Stepwise loss of sister chromatid cohesion ......... 15
1.3  Regulation of the prophase I-to-metaphase I transition
     in meiosis ................................................ 16
     1.3.1  Entry into metaphase I is regulated by the
            recombination checkpoint ........................... 16
     1.3.2  Meiosis-specific APC/C regulation during the
            prophase I-to-metaphase transition ................. 16
1.4  Aims of this study ........................................ 19
1.5  Contributions ............................................. 19

2    Results ................................................... 20
2.1  APC/C-Ama1 regulates the timing of the accumulation of
     Plk1 (Cdc5) ............................................... 20
2.2  Premature accumulation of Cdc5 in ama1Δ cells is
     partially rescued by the elimination of Nddl .............. 23
2.3  Premature accumulation of Cdc5 causes missegregation of
     homologous chromosomes during meiosis I ................... 24
2.4  Accumulation of Cdc5 and SC disassembly in ama1Δ cells
     does not depend on Ndt80 .................................. 25
2.5  Accumulation of Cdc5 and SC disassembly in ndt80Δ
     ama1Δ cells depend on Nddl ................................ 28
2.6  Accumulation of Cdc5 in ndt80Δ ama1Δ cells affects the
     synapsis initiation (ZMM) complex ......................... 31
2.7  Zip1 is degraded upon accumulation of Cdc5 even if it
     cannot bind to chromatin .................................. 34
2.8  Inhibition of Cdc5-as restores accumulation of Red1 and
     Zip1 in ndt80Δ ama1Δ cells ................................ 35
2.9  Premature accumulation of kinase activity of Cdc5 in
     ama1Δ cells results in inefficient formation of the
     synaptonemal complex ...................................... 37
2.10 Partial inhibition of Cdc5 rescues segregation of
     homologous chromosomes during meiosis I ................... 39
2.11 Rad52 as a marker for DSBs visualization in vivo .......... 41
2.12 Premature accumulation of Cdc5 results in entry into
     metaphase I in the presence of DNA double strand breaks ... 42
2.13 SC can be destroyed and re-assembled de novo in prophase
     upon induction and subsequent inhibition of Cdc5 .......... 43
2.14 SC can be formed during metaphase I upon inhibition of
     Cdc5 ...................................................... 46
2.15 Recombination checkpoint can be inhibited and
     re-activated upon induction followed by inhibition of
     Cdc5 in the presence of DSBs during metaphase I ........... 49

3    Discussion ................................................ 52
3.1  APC/C-Ama1 activity prevents premature accumulation of
     Cdc5 ...................................................... 52
3.2  Cdc5 induces disassembly of the SC on multiple levels ..... 53
3.3  How does Cdc5 regulate the degradation of SC components? .. 54
3.4  Implications for the function of the recombination
     checkpoint ................................................ 56
3.5  Concluding remarks ........................................ 58

4    Materials and methods ..................................... 59
4.1  Saccharomyces cerevisiae strains .......................... 59
4.2  Construction of yeast strains ............................. 59
4.3  Induction of synchronous meiosis .......................... 60
4.4  Microscopy ................................................ 61
     4.4.1  Indirect immunofluorescence microscopy of fixed
            cells .............................................. 61
     4.4.2  Indirect immunofluorescence microscopy of
            chromosome spreads ................................. 61
4.5  Live-cell imaging of meiosis .............................. 62
     4.5.1  Experimental setup ................................. 62
     4.5.2  Data presentation and analysis ..................... 63
4.6  Analysis of proteins ...................................... 63
     4.6.1  Preparation of protein extracts .................... 63
     4.6.2  SDS-PAGE and Western blotting ...................... 64

Abbreviations .................................................. 68
References ..................................................... 69
Acknowledgements ............................................... 81
Declaration .................................................... 82


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