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 14.2.2008 Table of Contents i Table of Contents Index of Figures ................................................................................................................v Index of Tables ...............................................................................................................vii 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 MRN Complex 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 Crystal 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 Data Collection and Structure 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 -Mre11................................................................85 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 Index of Figures Figure 1: Eukaryotic double-strand break repair pathways. ............................................. 8 Figure 2: Double-strand break repair through non-homologous end joining. ................ 10 Figure 3: Tethering of broken DNA molecules by the Rad50/Mre11 complex. ............ 12 Figure 4: Functional domains