H2A.Z-dependent cellular responses to a persistent DNA double-strand break [Elektronische Ressource] / vorgelegt von Natalie Jasmin Hiller

Natalie Hiller

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2010
• Nombre de lectures: 42
• Langue: Deutsch
• Poids de l'ouvrage: 27 Mo

Extrait

H2A.Z-Dependent Cellular Responses to a
Persistent DNA Double-Strand Break








Dissertation zur Erlangung des Doktorgrades der
Fakultät für Biologie der Ludwig-Maximilians-Universität München











vorgelegt von
Diplom-Biochemikerin
Natalie Jasmin Hiller







6. Mai 2010
Ehrenwörtliche Erklärung
Hiermit erkläre ich, dass ich die vorliegende Dissertation selbstständig 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.



München, den ............................. ............................................................
(Unterschrift)



















Promotionsgesuch eingereicht am: 6. Mai 2010
Datum der mündlichen Prüfung: 16. Juni 2010
Erster Gutachter: Prof. Dr. Stefan Jentsch
Zweiter Gutachter: Prof. Dr. Peter Becker


Die vorliegende Arbeit wurde zwischen September 2006 und Mai 2010 unter der
Anleitung von Prof. Dr. Stefan Jentsch am Max-Planck-Institut fur Biochemie
in Martinsried durchgefuhrt.

Wesentliche Teile dieser Arbeit sind in der folgenden Publikation veröffentlicht:



Kalocsay, M.*, Hiller, N. J.* & Jentsch, S. Chromosome-Wide Rad51
Spreading and SUMO-H2A.Z-Dependent Chromosome Fixation in Response
to a Persistent DNA Double-Strand Break. Molecular Cell 33, 335-43 (2009).

* these authors contributed equally to this work.










Note on results obtained in collaboration:
For the statistical analysis in figures 7A, 11, 12 and 15, data from experimental
repetitions performed by M. Kalocsay and N. Hiller in collaboration were used.
Experiments solely done by M. Kalocsay are not shown in figures here but
referenced by (Kalocsay, 2010; Kalocsay et al., 2009) when mentioned in the text.

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Dedicated to my parents




TABLE OF CONTENTS
SUMMARY...................................................................................................................1
1 INTRODUCTION ....................................................................................................2
1.1 Chromatin structure and function..2
1.1.1 Basic organization of chromatin............................................... 2
1.1.2 Chromatin dynamics ................................................................. 3
1.2 Sister chromatid cohesion.............11
1.2.1 The cohesin complex............................... 11
1.2.2 Establishment of sister chromatid cohesion .......................................................... 12
1.2.3 Cohesion establishment in response to DSBs........................ 14
1.3 DNA damage and repair ..............................................15
1.3.1 Repair of double-strand breaks by homologous recombination....................... 15
1.3.2 The DNA damage checkpoint............................................... 18
1.3.3 Adaptation to DNA damage.................................................. 20
1.3.4 DNA repair in the context of chromatin ................................................................ 20
1.4 Nuclear compartmentalization....23
2 AIM OF THIS STUDY.............................................................................................25
3 RESULTS ...............................................................................................................26
3.1 H2A.Z directs DSB processing and DNA damage checkpoint activation26
3.1.1 H2A.Z is implicated in DSB repair ............................................................................ 26
3.1.2 H2A.Z is required for proper resection of DSB ends.............. 27
3.1.3 H2A.Z is required for proper DNA damage checkpoint activation.................... 29
3.2 Role of H2A.Z in DSB repair ............................................................................30
3.3 A persistent DSB relocalizes to the nuclear envelope33
3.3.1 DSB movement to the nuclear envelope can be visualized in vivo .................. 33
3.3.2 Nuclear envelope protein Mps3 binds to the persistent DSB .............................. 34
3.3.3 DSB tethering requires H2A.Z, Rad51, and the DNA damage checkpoint. ...... 35
3.4 H2A.Z SUMOylation is required for DSB relocalization ................................37
3.5 Interactors of Mps3 at the nuclear envelope..............39
3.5.1 Mps3 binds to H2A.Z ................................................................. 39
3.5.2 Mps3 binds to DSB repair factors............................................ 42
3.5.3 Dissecting the function of the Mps3 nucleoplasmic domain.............................. 42
3.6 Possible functions of DSB relocation to the nuclear envelope ..................46
3.6.1 The fixed DSB end does not acquire telomere-like features ............................... 46
3.6.2 Mps3, DSB tethering and adaptation .................................................................... 47
3.7 Cohesion establishment in response to DSBs..............................................52
3.7.1 H2A.Z binds Eco1, the key player in cohesion establishment ............................. 52
3.7.2 H2A.Z-SUMOylation represses cohesion establishment........ 56
3.7.3 H2A.Z is required for Eco1-mediated cohesion at DSBs....................................... 60
4 DISCUSSION .......................................................................62
4.1 H2A.Z directs DSB-resection, checkpoint activation & repair ...................62
4.2 A persistent DSB relocalizes to the nuclear periphery................................66
4.2.1 Mechanism of break relocation to the nuclear periphery.. 66
4.2.2 Possible functions of break anchoring at the nuclear periphery ....................... 69
4.3 H2A.Z and sister chromatid cohesion ..........................................................72
5 MATERIALS AND METHODS................................................77
5.1 Microbiology ...................................................................77
5.1.1 Escherichia coli techniques........................................................ 77
5.1.2 Saccharomyces cerevisiae techniques....................................... 78
5.2 Molecular biological techniques...................................85
5.3 Biochemistry techniques ...............................................87
5.3.1 Protein methods ......................................... 87
5.3.2 Chromatin methods.................................................................... 91
5.4 Cell biological techniques .............................................97
5.4.1 Live-cell microscopy... 97
5.4.2 Cohesion assays......................................................................................................... 97
5.5 Computer-aided analysis.............98
6 REFERENCES........................................................................................................99
7 ABBREVIATIONS...............................117
8 ACKNOWLEDGEMENTS....................................................................................122
9 CURRICULUM VITAE.........................123


SUMMARY
DNA double-strand breaks (DSB) pose an extreme threat to genome stability.
Nevertheless, they occur frequently, being inflicted by γ-irradiation and certain
genotoxins, but also arising sporadically during faulty replication. If left unrepaired,
DSBs can cause chromosome-loss-associated lethality or translocation-driven
tumorigenesis. To overcome this fundamental, genotoxic insult, cells have evolved
elaborate DNA repair systems, most importantly, homologous recombination (HR),
which needs homologous sequences to guide repair and non -homologous end-
joining (NHEJ), which involves ligation of DSB ends and is error-prone. By default,
DSB repair must function in the context of chromatin. Only recently it is appreciated,
how repair pathways have in fact harnessed the preexisting, vast regulatory potential
of epigenetics to fine-tune and diversify the cellular DNA damage response (DDR).
This study identified the histone-variant H2A.Z as an important new and early
factor in the DDR, being positioned at the vertex of DSB-processing and DNA
damage checkpoint activation. Mutants in the gene for H2A.Z are severely sensitive
to DSB-inducing agents and defective in both DSB-resection and DSB repair via
single-strand annealing. Research over