Characterisation of CHRAC14 and CHRAC16, the two histone fold subunits of the chromatin accessibility complex [Elektronische Ressource] / Klaus Felix Hartlepp

Klaus Felix Hartlepp

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2006
• Nombre de lectures: 17
• Langue: English
• Poids de l'ouvrage: 17 Mo

Extrait

Dissertation zur Erlangung des Doktorgrades
der Fakultät für Chemie und Pharmazie
der Ludwig-Maximilians-Universität München
Characterisation of CHRAC14 and CHRAC16,
the two Histone Fold Subunits of the
Chromatin Accessibility Complex
Klaus Felix Hartlepp
aus
Augsburg
2006Erklärung
Diese Dissertation wurde im Sinne von § 13 Abs. 3 bzw. 4 der Promotionsordnung vom
29. Januar 1998 von Herrn Prof. Dr. Peter Becker betreut und von Herrn Prof. Dr. Patrick
Cramer vor der Fakultät für Chemie und Pharmazie vertreten.
Ehrenwörtliche Versicherung
Diese Dissertation wurde selbständig, ohne unerlaubte Hilfe erarbeitet.
München, am 23. Januar 2006
.......................................................................
K. Felix Hartlepp
Dissertation eingereicht am 27.01.2006
1. Gutachter Prof. Dr. Peter Becker
2. Gutachter Prof. Dr. Patrick Cramer
Mündliche Prüfung am 22.03.2006To make them run easily and swiftly, the axles of carriages are anointed;
and for much the same purpose, some whalers perform an analogous
operation upon their boat; they grease the bottom. Nor is it to be doubted
that as such a procedure can do no harm, it may possibly be of no
contemptible advantage; considering that oil and water are hostile; that oil is
a sliding thing, and that the object in view is to make the boat slide bravely.
Herman Melville – Moby Dick, 1851Acknowledgements
Here, I would like to express my gratitude to everybody who supported me during my PhD thesis.
First of all, I would like to thank my supervisor Prof. Peter Becker for giving me the opportunity to
work on an exciting project in an excellent environment. Peter’s constant support, his encouragement
and expertise have been very inspiring and motivating and were especially helpful to me in the most
challenging situations.
I am also deeply grateful to Dr. Anton Eberharter for teaching me several techniques that have been
crucial for this work, discussing my results and giving me valuable advise throughout my thesis,
providing me with nucleosomes, baculoviruses, and other materials, proof-reading of this manuscript
and his Austrian humor.
I am much obliged to the fruitful collaboration with Drs. Christoph Müller, Carlos Fernández-Tornero
and Tim Grüne (EMBL Grenoble Outstation), who did not only perform the crystal structure analysis
in this work, but also instructed me in protein crystallisation and introduced me to the basics of
structure determination.
Moreover, I thank Dr. Elisabeth Kremmer and her colleagues (GSF, Munich) for producing and
screening the α-CHRAC14/ α-CHRAC16 rat monoclonal antibodies, Norbert Mücke and Prof. Jörg
Langowski (DKFZ, Heidelberg) for performing analytical ultracentrifugation studies, and Dr. Axel
Imhof, Dr. Lars Israel and Tilman Schlunck (Zentrallabor für Proteinanalytik, ABI, Munich) for mass
spectrometry analysis.
I would also like to thank all the people working at the ABI department of molecular biology for
permanent help and advise in daily lab life and for creating a great atmosphere. It is a pleasure to work
with you!
Special thanks go to my parents Ingrid and Werner Hartlepp, who strongly supported me both
financially and by constant motivation during the past decade of my studies and thereby contributed
fundamentally to this work.
Finally, I thank Hubert Kettenberger, not only for endless discussions about my work, for sharing his
knowledge of structural biology, chemistry and transcription, for proof-reading of this manuscript and
for his formatting skills, but also for being there.TABLE OF CONTENTS i
Table of contents
1 Summary .................................................................................................................. 1
2 Introduction ............................................................................................................. 2
2.1 Chromatin structure............................................................................................................. 2
2.1.1 The nucleosome............................................................................................................... 2
2.1.2 Higher order structures of chromatin........................................................................... 3
2.2 The histone fold....... 5
2.2.1 Structure of the core histones ........................................................................................ 5
2.2.2 Histone fold proteins ......................................................................................................6
2.2.2.1 Archeal histone fold proteins .................................................................................... 6
2.2.2.2 TATA box-binding protein-associated factors (TAFs) ......................................... 7
2.2.2.3 Negative Cofactor 2 (NC-2) ...................................................................................... 9
2.2.2.4 Nuclear Factor Y (NF-Y)........................................................................................... 9
2.2.2.5 Subunits of DNA Polymerase epsilon ...................................................................10
2.2.2.6 Subunits of the Chromatin Accessibility Complex (CHRAC)............................10
2.3 Chromatin dynamics and regulation................................................................................11
2.3.1 Histone modifications...................................................................................................11
2.3.2 Histone variants .............................................................................................................12
2.3.3 Histone chaperones.......................................................................................................13
2.3.4 HMG box proteins........................................................................................................14
2.3.5 ATP-dependent chromatin remodelling complexes.................................................15
2.3.5.1 SWI/SNF complexes................................................................................................17
2.3.5.2 ISWI complexes17
2.3.5.3 CHD1/Mi-2 complexes ...........................................................................................22
2.3.5.4 INO80/SWR1 complexes........................................................................................22
2.4 Insights into structure and mechanism of ATP-dependent
chromatin remodelling.......................................................................................................23
2.4.1 A variety of remodelling scenarios ..............................................................................23
2.4.2 Mechanisms of chromatin remodelling: DNA-twisting or DNA-bulging? ..........24
2.4.3 The Rad54 ATPase domain .........................................................................................26
2.4.4 SWI/SNF complexes ....................................................................................................28
2.4.5 ISWI-containing complexes .........................................................................................29
2.4.5.1 ISWI complexes in Drosophila...............................................................................29
2.4.5.2 ISW2............................................................................................................................31
2.5 The Chromatin Accessibility Complex (CHRAC).........................................................33TABLE OF CONTENTS ii
3 Materials and methods........................................................................................... 35
3.1 Materials...............................................................................................................................35
3.1.1 Solutions, buffers and media........................................................................................35
3.1.2 Organisms, cells and strains .........................................................................................37
3.1.2.1 E. coli strains..............................................................................................................37
3.1.2.2 Insect cells and fly lines ............................................................................................37
3.1.2.3 Baculoviral expression vectors ................................................................................37
3.1.3 Vectors, plasmids and oligonucleotides......................................................................38
3.1.3.1 Vectors and plasmids................................................................................................38
3.1.3.2 Oligonucleotides........................................................................................................40
3.1.3.3 Antibodies ..................................................................................................................45
3.2 Methods ...............................................................................................................................47
3.2.1 Cloning of expression vectors......................................................................................47
3.2.1.1 Recombinant CHRAC14-CHRAC16 in E. coli....................................................47
3.2.1.2 In vitro translation of ACF1 deletion constructs .................................................47
3.2.2 Site-directed