Structural characterization of the DNA repair protein complex SbcC-SbcD of Thermotoga maritima [Elektronische Ressource] / Derk Julius Bemeleit

Derk Bemeleit

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2008
• Nombre de lectures: 39
• Langue: Deutsch
• Poids de l'ouvrage: 5 Mo

Extrait

Dissertation zur Erlangung des Doktorgrades
der Fakultät für Chemie und Pharmazie
der Ludwig-Maximilians-Universität München






Structural Characterization
of the DNA Repair Protein Complex
SbcC-SbcD
of Thermotoga maritima














Derk Julius Bemeleit

aus

Bremen



München, 2008 Erklärung
Diese Dissertation wurde im Sinne von § 13 Abs. 3 der Promotionsordnung vom 29.
Januar 1998 von Herrn Prof. Dr. Karl-Peter Hopfner betreut.



Ehrenwörtliche Versicherung
Diese Dissertation wurde selbständig, ohne unerlaubte Hilfsmittel erarbeitet.

München, am ..............

...........................
Derk Bemeleit








Dissertation eingereicht am ...................................
1. Gutachter: Herr Prof. Dr. Karl-Peter Hopfner
2. Gutachter: Herr Prof. Dr. Patrick Cramer
Mündliche Prüfung am 14.2.2008Table of Contents i
Table of Contents
Index of Figures ................................................................................................................v
Index of Tablesvii
1 Summary......................................................................................................................1
2 Introduction..................................................................................................................2
2.1 DNA Damage and Repair .....................................................................................2
2.1.1 DNA Damages.............................................................................................2
2.1.1.1 Endogenous DNA Damages..........................................................3
2.1.1.2 Environmental DNA Damages......................................................4
2.1.1.3 Biologically Required Breaks.......................................................5
2.1.2 DNA Repair.................................................................................................6
2.1.2.1 DNA Double-Strand Break Repair................................................7
2.2 The Mre11 – Rad50 – Nbs1 complex .................................................................15
2.2.1 Structural and Biochemical Properties ......................................................16
2.2.2 Features of the Bacterial SbcCD (Rad50-Mre11) Complex......................20
2.2.3 Rad50 – Mre11 Interaction........................................................................24
2.2.4 ATM and Checkpoint Activation ..............................................................25
2.2.5 MRN Complex in Replication...................................................................26
2.2.6 in Telomere Maintenance ................................................27
2.2.7 MRN Complex and Diseases.....................................................................29
2.3 Structure Determination......................................................................................29
2.3.1 X-ray Crystallography...............................................................................30
2.3.2 Small Angle X-ray Scattering....................................................................34 Table of Contents ii
3 Objectives...................................................................................................................39
4 Materials.....................................................................................................................40
4.1 Chemicals............................................................................................................40
4.2 Molecular Biology Methods...............................................................................40
4.3 Enzymes, Standards and Chromatographic Material..........................................41
4.4 Oligonucleotides.................................................................................................42
4.5 E.coli strains and Plasmids..................................................................................43
5 Methods......................................................................................................................44
5.1 Cloning................................................................................................................44
5.2 Bioinformatics and Protein Parameters...............................................................47
5.3 Protein Expression..............................................................................................48
5.4 Protein Purification.............................................................................................50
5.5 SDS-PAGE..........................................................................................................52
5.6 Analytical Size Exclusion Chromatography.......................................................52
5.7 Electrophoretic Mobility Shift Assay .................................................................53
5.8 Anisotropy...........................................................................................................54
5.9 Crystallization.....................................................................................................55
5.10 X-ray Data Collection and Structure Determination...........................................56
5.11 SAXS Sample Preparation, Data Collection and Structure Determination ........57
5.12 Cry stal Staining ................................................................................................... 59
6 Results........................................................................................................................61
6.1 Cloning, Expression and Purification .................................................................61
6.1.1 Thermotoga maritima SbcCD ...................................................................61
6.1.2 Pyrococcus furiosus Rad50NCM11 ..........................................................63 Table of Contents iii
6.2 Biochemical Studies............................................................................................65
6.2.1 Electrophoretic Mobility Shift Assay........................................................65
6.2.2 Anisotropy.................................................................................................66
6.3 X-ray Crystallography.........................................................................................67
6.3.1 Crystallization............................................................................................67
6.3.2 Data Collection..........................................................................................69
6.3.3 Structure Determination and Refinement..................................................70
6.3.4 Crystallization and Data Collection of SbcC in Complex with DNA .......72
6.4 SAXS..................................................................................................................73
6.4.1 Data Collection and Structure Determination of the Apo SbcCD
Complex ....................................................................................................73
6.4.2 cture Determination of the Nucleotide-Bound
SbcCD Complex........................................................................................77
6.5 Structural Analysis..............................................................................................80
6.5.1 X-ray Crystallography...............................................................................80
6.5.1.1 Overall Structure of SbcCD ........................................................80
6.5.1.2 SbcD Interface.............................................................................82
6.5.1.3 SbcC-SbcD Interaction................................................................83
6.5.1.4 Comparison of Thermotoga maritima SbcCD with Pyrococcus
furiosus Rad50 -Mre1185
6.5.2 Small angle X-ray Scattering.....................................................................89
6.5.2.1 Small angle X-ray Scattering of the Apo SbcCD Complex ........89
6.5.2.2 Small angle X-ray Scattering of the Nucleotide-Bound SbcCD
Complex ......................................................................................90
7 Discussion..................................................................................................................92
8 Outlook.....................................................................................................................106 Table of Contents iv
9 References................................................................................................................108
10 Abbreviations ................................................................................................................I
Appendix.........................................................................................................................III
Acknowledgements......................................................................................................VIII
Curriculum vitae.............................................................................................................IX

Index of Figures v