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Informations
Publié par | ludwig-maximilians-universitat_munchen |
Publié le | 01 janvier 2010 |
Nombre de lectures | 14 |
Langue | English |
Poids de l'ouvrage | 19 Mo |
Extrait
Dissertation zur Erlangung des Doktorgrades
der Fakultät für Chemie und Pharmazie
der Ludwig-Maximilians-Universität München
Structures and DNA-Binding Activities of the
Hinge Domains from the Structural Maintenance
of Chromosomes Proteins of Pyrococcus furiosus
and the Mouse Condensin Complex
Julia Johanna Griese
aus
Erbach im Odenwald
2010
Erklärung
Diese Dissertation wurde im Sinne von § 13 Abs. 3 bzw. 4 der Promotionsordnung vom
29. Januar 1998 von Herrn Prof. Dr. Karl-Peter Hopfner betreut.
Ehrenwörtliche Versicherung
Diese Dissertation wurde selbständig, ohne unerlaubte Hilfe erarbeitet.
München, am 19.08.2010
…………………………………
Julia Johanna Griese
Dissertation eingereicht am 19.08.2010
1. Gutachter: Herr Prof. Dr. Karl-Peter Hopfner
2. Gutachter: Frau Prof. Dr. Elena Conti
Mündliche Prüfung am 25.10.2010
This thesis has been prepared from March 2007 to August 2010 in the laboratory of
Professor Dr. Karl-Peter Hopfner at the Gene Center of the Ludwig-Maximilians-
University of Munich (LMU).
Parts of this thesis have been published:
Griese, J.J., Witte, G. and Hopfner, K.P. (2010) Structure and DNA binding activity of the
mouse condensin hinge domain highlight common and diverse features of SMC proteins.
Nucleic Acids Res., 38, 3454-3465.
Griese, J.J. and Hopfner, K.P. (2010) Structure and DNA-binding activity of the
Pyrococcus furiosus SMC Protein Hinge Domain. Proteins: Struct. Funct. Bioinform., in
press.
Parts of this thesis have been presented at international conferences and workshops:
ndPoster presentation and talk at the 2 EU-IP DNA Repair Workshop for Young Scientists,
June 23-27, 2008 in Porto, Portugal.
rdPoster presentation and talk at the 3 EU-IP DNA Repair Workshop for Young Scientists,
February 19-21, 2009 in Taormina, Sicily, Italy.
Poster presentation at the Gordon Research Conference on Diffraction Methods in
Structural Biology, July 18-23, 2010 in Lewiston, Maine, USA.
TABLE OF CONTENTS
TABLE OF CONTENTS
1 SUMMARY ........................................................................................................................ 1
2 INTRODUCTION ............... 2
2.1 The Discovery of Chromosomes ............................................................................ 2
2.2 Structural Maintenance of Chromosomes Proteins ............ 3
2.2.1 Molecular Architecture of SMC Proteins and SMC Complexes ....................... 4
2.2.1.1 The SMC Head Domain ....................................................................................................... 7
2.2.1.2 The SMC Hinge Domain ..... 9
2.2.2 The Function and Mechanism of Cohesin ....................... 10
2.2.2.1 Cohesin Function in Mitosis and Meiosis ......................................................................... 10
2.2.2.2 The Molecular Mechanism of Cohesin .............. 12
2.2.2.3 Cohesin Function in DNA Repair ...................................................................................... 13
2.2.3 The Function and Mechanism of Prokaryotic and Eukaryotic Condensins .... 14
2.2.3.1 Condensin Function in Mitosis .......................... 14
2.2.3.2 The Molecular Mechanism of Condensin .......................................................................... 16
2.2.3.3 Condensin Function in DNA Repair .................. 17
2.2.4 The Function and Mechanism of the SMC5-SMC6 Complex ........................ 18
2.2.5 The DNA-Loading Mechanism of SMC Complexes ...................................... 19
2.3 Objectives .............................................................................. 21
3 MATERIALS AND METHODS .......................................................... 23
3.1 Materials ................ 23
3.2 Molecular Biology Methods ................................................................................. 23
3.2.1 Cloning and Site-Directed Mutagenesis .......................... 23
3.3 Microbiology Methods ......................... 26
3.3.1 Transformation of E. coli ................................................................................ 26
3.3.2 Recombinant Protein Production in E. coli ..................... 27
3.4 Protein Biochemistry Methods ............ 29
3.4.1 Purification of Recombinant Proteins ............................................................. 29
3.4.2 Denaturing Polyacrylamide Gel Electrophoresis (SDS-PAGE) ...................... 30
3.4.3 Analytical Size Exclusion Chromatography ................................................... 31
3.4.4 Dynamic Light Scattering Analysis ................................. 31
3.5 Structural Biology Methods ................................................. 31
3.5.1 Background ...................................... 31
i TABLE OF CONTENTS
3.5.2 X-ray Crystallography ..................................................................................... 32
3.5.2.1 Crystallisation ................................................... 32
3.5.2.2 Data Collection ................................................. 33
3.5.2.3 Structure Determination, Model Building and Refinement ............... 33
3.5.3 Small-Angle X-ray Scattering of Protein Solutions ........ 34
3.5.3.1 Sample Preparation ........................................................................................................... 34
3.5.3.2 Data Collection, Processing and Analysis ........ 34
3.6 In Vitro DNA-Binding Assays .............................................................................. 35
3.6.1 Preparation of DNA Substrates ....... 35
3.6.2 Electrophoretic Mobility Shift Assays ............................................................ 36
3.6.3 Fluorescence Quenching Titrations ................................. 37
4 RESULTS ........................................................................................................................ 39
4.1 Crystal and Solution Structures of SMC Hinge Domains 39
4.1.1 The Pyrococcus furiosus SMC Hinge Domain ............................................... 39
4.1.1.1 Cloning, Purification and Biochemical Characterisation ................. 39
4.1.1.2 Crystallisation and Structure Determination .................................... 40
4.1.1.3 Crystal Structure of the P. furiosus SMC Hinge Domain .................. 43
4.1.1.4 Similarity Between the P. furiosus and Other Prokaryotic SMC Hinge Domains ............. 46
4.1.1.5 Solution Scattering Analysis of the P. furiosus SMC Hinge Domain ................................. 48
4.1.2 The Mouse Condensin Hinge Domain ............................................................ 49
4.1.2.1 Cloning, Purification and Biochemical Characterisation ................................................. 49
4.1.2.2 Crystallisation and Structure Determination .... 53
4.1.2.3 Crystal Structure of the Mouse Condensin Hinge Domain ............... 55
4.1.2.4 Analysis of the SMC2-SMC4 Hinge Domain Interface ..................................................... 59
4.1.2.5 Solution Scattering Analysis of the Mouse Condensin Hinge Domain .............................. 60
4.2 DNA-Binding Activity of SMC Hinge Domains................. 62
4.2.1 DNA-Binding Activity of the Mouse Condensin Hinge Domain ................... 62
4.2.1.1 Electrophoretic Mobility Shift Assays ............................................................................... 63
4.2.1.2 Quantitative Fluorescence Quenching Titrations .............................................................. 65
4.2.1.3 DNA-Binding Activity of Lysine-to-Glutamate Point Mutants .......... 67
4.2.2 DNA-Binding Activity of the P. furiosus SMC Hinge Domain ..................... 70
4.2.2.1 Electrophoretic Mobility Shift Assays ............................................................................... 70
4.2.2.2 DNA-Binding Activity of Lysine-to-Glutamate Point Mutants .......... 72
5 DISCUSSION ................................................................................................................... 75
5.1 The SMC Hinge Domain Fold is Highly Conserved .......... 75
ii TABLE OF CONTENTS
5.2 Condensin SMC Hinge Domains Preferentially Bind Single-Stranded DNA . 78
5.2.1 Localisation of the DNA-Binding Surface ...................................................... 78
5.2.2 Functional Implications of the Single-Stranded DNA-Binding Activity ........ 81
5.2.3 Functional Implications of the Double-Stranded DNA-Binding Activity ...... 82
5.3 Conclusion ............................................................................................................. 83
6 REFERENCES ....................I
7 APPENDIX .................................................................................................................... XX
7.1 The Bicistronic Vector for Heterodimeric Expression Constructs ................ XX
7.2 Amino Acid Sequences and Physico-Chemical Parameters of Proteins ....... XXI
7.3 Abbreviations ................................................................................