Coordination of synaptonemal complex formation and pachytene checkpoint signaling in meiosis [Elektronische Ressource] / vorgelegt von Christian Eichinger

Christian Eichinger

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2009
• Nombre de lectures: 19
• Langue: Deutsch
• Poids de l'ouvrage: 4 Mo

Extrait

Coordination of synaptonemal complex
formation and pachytene checkpoint
signaling in meiosis





Dissertation der
Fakultät für Biologie der
Ludwig-Maximilians-Universität
München




vorgelegt von
Diplom-Biologe
Christian Stefan Eichinger


4. Mai 2009






























Hiermit erkläre ich, dass ich die vorliegende Dissertation selbständig und
ohne unerlaubte Hilfe angefertigt habe. Ich habe weder anderweitig versucht,
eine Dissertation einzureichen oder eine Doktorprüfung durchzuführen, noch
habe ich diese Dissertation oder Teile derselben einer anderen
Prüfungskommission vorgelegt.



Christian Eichinger
München, den 4. Mai 2009

















Promotionsgesuch eingereicht: 4. Mai 2009
Tag der mündlichen Prüfung: 14. Juli 2009
Erster Gutachter: Prof. Dr. Stefan Jentsch
Zweiter Gutachter: Prof. Dr. Peter Becker
Die vorliegende Arbeit wurde zwischen April 2005 und April 2009 unter der
Anleitung von Prof. Dr. Stefan Jentsch am Max-Planck-Institut für Biochemie
in Martinsried durchgeführt.

Wesentliche Teile dieser Arbeit sind in der folgenden Publikation
veröffentlicht:

Christian S. Eichinger & Stefan Jentsch. Synaptonemal complex formation
and pachytene checkpoint signaling are coordinated through Red1!9-1-1
interaction (submitted to Nature Cell Biology, 2009).



















































Table of contents
I SUMMARY..........................................................................................................................1
II INTRODUCTION..............................................................................................................2
II.1 Ubiquitin and ubiquitin-like modifiers.............................................................................2
II.1.1 The ubiquitin and SUMO conjugation system...................................................................3
II.1.2 Functions of ubiquitin and SUMO.....................................................................................6
II.2 Meiosis..............................................................................................................................11
II.2.1 Major processes in meiosis.............................................................................................11
II.2.2 Role of SUMO in meiosis................................................................................................14
II.3 DNA damage response...................................................................................................15
II.3.1 DNA repair pathways......................................................................................................15
II.3.2 DNA damage checkpoints..............................................................................................16
II.3.3 The 9-1-1 checkpoint complex........................................................................................16
II.3.4 Meiotic recombination and surveillance mechanisms…………………............................18
II.4 Aim....................................................................................................................................24
III RESULTS........................................................................................................................25
III.1 Red1 is modified by SUMO during meiosis.................................................................25
III.1.1 Purification of meiotic SUMO substrates in S. cerevisiae..............................................25
III.1.2 The SUMO substrate Red1............................................................................................25
III.1.3 The SUMO acceptor lysines in Red1.............................................................................26
III.1.4 The SUMO substrate Sycp3 in Homo sapiens..............................................................28
III.2 Red1 SUMOylation recruits Zip1 for timely establishment of SCs……………...……29
III.2.1 Red1 and Zip1 interact in a SUMO-dependent manner.................................................29
III.2.2 Red1 SUMO-deficient mutant shows delayed zipping…………………………………….31
III.3 Red1 binds 9-1-1 for pachytene checkpoint activation and normal SC formation..35
III.3.1 Domain mapping and identification of specific point mutants........................................35
III.3.2 Function of Red1 interaction with the 9-1-1 complex……………………………………...37
III.4 Regulation of Red1 SUMOylation and expression......................................................41
III.4.1 Regulation of Red1 SUMOylation..................................................................................41
III.4.2 Regulation of Red1 expression……………………………..............................................44
III.5 SUMOylation of the 9-1-1 complex................................................................................46
III.5.1 Each 9-1-1 subunit is modified with SUMO...................................................................46
III.5.2 Regulation of 9-1-1 SUMOylation..................................................................................48
III.5.3 Towards a function for the SUMOylation of 9-1-1..........................................................49
III. 6 Psy2 links the SC to pachytene checkpoint exit………….…………………………….51
III. 6.1 Interaction between Zip1 and Psy2...............................................................................51
III. 6.2 Psy2 functions in meiotic checkpoint control................................................................54
IV DISCUSSION ................................................................................................................60
IV.1 Red1 SUMOylation is important for timely zipping……………………………………..67
Table of contents
IV.2 Red1 binds 9-1-1 for pachytene checkpoint activation and normal SC formation..67
IV.3 Intimate connection between pachytene checkpoint and SC formation……………70
V MATERIAL AND METHODS.....................................................................................74
V.1 Computational analyses.................................................................................................74
V.2 Microbiological and genetic techniques ......................................................................74
V.2.1 Escherichia coli techniques............................................................................................74
V.2.2 Saccharomyces cerevisiae techniques..........................................................................76
V.3 Cell biological techniques..............................................................................................82
V.3.1 Tissue culture……………...........…………………………………………………………….82
V.3.2 Live-cell microscopy…………………………………………………………………………..83
V.4 Molecular biology techniques........................................................................................83
V.4.1 Isolation of DNA..............................................................................................................84
V.4.2 Polymerase chain reaction (PCR)..................................................................................85
V.4.3 Cloning of plasmids constructs.......................................................................................88
V.5 Biochemical techniques.................................................................................................89
V.5.1 Gel electrophoresis and immunoblot techniques............................................................89
V.5.2 Preparation of cell extracts.............................................................................................92
V.5.3 Protein purification and binding experiments……………………………………………….93
VI REFERENCES..............................................................................................................96
ABBREVIATIONS……………………………………………………………………………………….
ACKNOWLEDGEMENTS…….....................................................................................................
CURRICULUM VITAE.................................................................................................................
Summary
I SUMMARY

Genome integrity is challenged by numerous endogenous and exogenous
DNA damages which are potentially harmful for an organism. In order to
preserve the genetic information, errors in the DNA have to be rapidly
recognized and repaired. Therefore, cells are equipped with two highly
interconnected mechanisms: DNA damage checkpoints that alert and signal
the presence of DNA mistakes and DNA repair