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Structural and Functional Analysis of the Eukaryotic DNA Repair Proteins Mre11 and Nbs1

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Structural and Functional Analysis of the Eukaryotic DNA Repair Proteins Mre11 and Nbs1 Dissertation zur Erlangung des Doktorgrades der Fakultät für Chemie und Pharmazie der Ludwig-Maximilians-Universität München Structural and functional analysis of the eukaryotic DNA repair proteins Mre11 and Nbs1 Christian Bernd Schiller aus Kassel 2011 Erklärung Diese Dissertation wurde im Sinne von § 13 Abs. 3 bzw. 4 der Promotionsordnung vom 29. Januar 1998 (in der Fassung der sechsten Änderungssatzung vom 16. August 2010) von Herrn Prof. Dr. Karl-Peter Hopfner betreut. Ehrenwörtliche Versicherung Diese Dissertation wurde selbständig, ohne unerlaubte Hilfe erarbeitet. München, am 07.06.2011 .................................................... (Christian Bernd Schiller) Dissertation eingereicht am 07.06.2011 1. Gutachter: Herr Prof. Dr. Karl-Peter Hopfner 2. Gutachter: Herr Prof. Dr. Dietmar Martin Mündliche Prüfung am 21.07.2011 During the work of this thesis, the following publication was published: Lammens K., Bemeleit D. J., Möckel C., Clausing E., Schele A., Hartung S., Schiller C. B., Lucas M., Angermüller C., Soding J., Strässer K. and K. P. Hopfner (2011). "The Mre11:Rad50 Structure Shows an ATP-Dependent Molecular Clamp in DNA Double-Strand Break Repair." Cell 145(1): 54-66. Parts of the present thesis will be submitted for publication: Schiller C.B., Lammens K., Guerini I., Coordes B., Schlauderer F., Möckel C., Schele A., Sträßer K., Jackson S. P., Hopfner K.-P.: “Insights into DNA double-strand break repair and ataxia-telangiectasia like disease from the structure of an Mre11-Nbs1 complex“, manuscript in preparation. Parts of this thesis have been presented at international conferences and workshops: Talk and poster at the Biannual International Meeting of the German Society of DNA Repair Research (DGDR) - Repair meets Replication, September 7-10, 2010 in Jena, Germany Poster presentation at the Gordon Research Conference on Mutagenesis - Consequences of Mutation and Repair for Human Disease, August 1-6, 2010 in Waterville, Maine, USA. Poster presentation at the 2nd EU-IP DNA Repair Workshop for Young Scientists, June 23-27, 2008 in Porto, Portugal. Poster presentation at the 1st EU-IP DNA Repair Workshop for Young Scientists, May 13-16, 2007, 2007 in Gent, Belgium. TABLE OF CONTENTS TABLE OF CONTENTS 1. SUMMARY ........................................................................................................................... 1 2. INTRODUCTION ................................................................................................................... 2 2.1 Biological roles of DNA double-strand breaks ............................................................ 2 2.1.1 DNA double strand breaks in cellular metabolism processes ........................................................... 2 2.1.2 Environmentally caused DNA double strand breaks ........................................................................ 3 2.2 DNA double-strand repair pathways - A short overview ............................................. 5 2.3 The Mre11-Rad50-Nbs1 complex - biochemistry and structural architecture............. 7 2.3.1 Biochemical in vitro activities of Mre11-Rad50-Nbs1 ..................................................................... 8 2.3.2 Structural architecture of the Mre11-Rad50-Nbs1 complex ............................................................. 9 2.4 The Mre11-Rad50-Nbs1 complex in double-strand break repair .............................. 13 2.4.1 The Mre11-Rad50-Nbs1 complex in homologous recombination.................................................. 13 2.4.2 The Mre11-Rad50-Nbs1 complex in meiotic recombination ......................................................... 15 2.4.3 The Mre11-Rad50-Nbs1 complex in telomere maintenance .......................................................... 16 2.4.4 The Mre11-Rad50-Nbs1 complex in non-homologous end joining pathways ............................... 17 2.5 The Mre11-Rad50-Nbs1 complex in DNA damage signaling ................................... 18 2.6 Diseases linked with mutations in Mre11-Rad50-Nbs1............................................. 20 2.7 Objectives ................................................................................................................... 22 3. MATERIALS AND METHODS .............................................................................................. 23 3.1 Materials ..................................................................................................................... 23 3.1.1 Antibodies....................................................................................................................................... 23 3.1.2 Oligonucleotides ............................................................................................................................. 24 3.1.3 Plasmids .......................................................................................................................................... 27 3.1.4 Strains ............................................................................................................................................. 29 3.2 Media and antibiotics ................................................................................................. 30 3.3 Methods ...................................................................................................................... 31 3.3.1 Molecular biology methods ............................................................................................................ 31 3.3.1.1 Molecular cloning ..................................................................................................................... 31 3.3.1.2 Site Directed Mutagenesis by Overlap Extension PCR............................................................. 32 3.3.1.3 Transformation in E. coli .......................................................................................................... 33 3.3.2 Protein biochemistry methods ........................................................................................................ 33 3.3.2.1 Protein expression in E. coli ..................................................................................................... 33 TABLE OF CONTENTS 3.3.2.2 Recombinant selenomethionine expression in E. coli ............................................................... 33 3.3.2.3 Purification of GST-labelled proteins ....................................................................................... 34 3.3.2.4 Purification of His-tag labeled proteins ................................................................................... 35 3.3.2.5 Discontinous Polyacrylamide Gel Electrophoresis (SDS-PAGE) ............................................ 36 3.3.2.6 Western blot analysis ................................................................................................................ 37 3.3.2.7 Analytical size exclusion chromatography ................................................................................ 38 3.3.2.8 Limited Proteolysis ................................................................................................................... 38 3.3.2.9 Nuclease activity assay ............................................................................................................. 38 3.3.2.10 EMSA (electrophoretic mobility shift assay) ............................................................................. 39 3.3.3 Structural biology methods ............................................................................................................. 39 3.3.3.1 Crystallization ........................................................................................................................... 39 3.3.3.2 Data collection, structure solution and model building ............................................................ 40 3.3.3.3 Small angle x-ray scattering ..................................................................................................... 41 3.3.4 Yeast specific methods ................................................................................................................... 42 3.3.4.1 Yeast transformation ................................................................................................................. 42 3.3.4.2 Plate survival assays ................................................................................................................. 42 3.3.4.3 Co-immunoprecipitation ........................................................................................................... 42 3.3.4.4 Indirect immunofluorescence .................................................................................................... 43 3.3.5 Bioinformatical methods ................................................................................................................ 44 3.3.5.1 Structure based sequence alignments ....................................................................................... 44 4. RESULTS ............................................................................................................................ 46 4.1 Cloning and expression of Mre11 and Nbs1 from S. pombe ..................................... 46 4.2 Crystallization, structure solution and refinement ..................................................... 49 4.2.1 Nbs1mir-Mre11cd complex ............................................................................................................... 49 4.2.2 Apo-Mre11cd ..................................................................................................................................
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