Structural and functional analysis of ATP dependent conformational changes in the bacterial Mre11:Rad50 catalytic head complex [Elektronische Ressource] / Carolin Möckel. Betreuer: Karl-Peter Hopfner

Carolin Möckel

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2011
• Nombre de lectures: 18
• Langue: English
• Poids de l'ouvrage: 13 Mo

Extrait

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







Structural and functional analysis of ATP dependent
conformational changes in the bacterial
Mre11:Rad50 catalytic head complex




Carolin Möckel
aus
München



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, den 19. Dezember 2011



.....................................................
Carolin Möckel







Dissertation eingereicht am 14. Oktober 2011
1. Gutachter: Prof. Dr. Karl-Peter Hopfner
2. Gutachter: Prof. Dr. Patrick Cramer
Mündliche Prüfung am 12. Dezember 2011


This thesis has been prepared from November 2007 to October 2011 in the laboratory of
Prof. Dr. Karl-Peter Hopfner at the Gene Center of the Ludwig-Maximilians-University of
Munich (LMU).



Publications

During the work of this thesis, the following publications have been published or are in the
process of being published:

* * *Lammens K. , Bemeleit D. J. , Möckel C. , Clausing E., Schele A., Hartung S., Schiller C.
B., Lucas M., Angermüller C., Söding J., Strässer K., and Hopfner K.-P. (2011). The
Mre11:Rad50 Structure Shows an ATP-Dependent Molecular Clamp in DNA Double-
Strand Break Repair. Cell 145(1): 54-66.

* These authors contributed equally to this work

Möckel C., Lammens K., Schele A., and Hopfner K.-P. (2011). ATP driven structural
changes of the bacterial Mre11:Rad50 catalytic head complex. Nucleic Acids Res. 2011
Sep 21. [Epub ahead of print]

Schiller C.B., Lammens K., Guerini I., Coordes B., Schlauderer F., Möckel C., Schele A.,
Sträßer K., Jackson S. P. and Hopfner K.-P. (2011). Insights into DNA double-strand
break signaling and ataxia-telangiectasia like disease from the structure of an Mre11-
Nbs1 complex. Manuscript submitted.
Table of Contents i

Table of Contents
1.  Summary .................................................................................................................................... 1 
2.  Introduction ................................................................................................................................ 2 
2.1.  DNA – history of the keystone of life ................................................................................ 2 
2.2.  DNA damage ...................................................................................................................... 2 
2.2.1.  DNA double strand breaks (DSBs) ............................................................................ 4 
2.3.  DSB repair ...... 6 
2.3.1.  Homologous recombination and single strand annealing ........................................... 8 
2.3.2.  Classical and alternative non-homologous end-joining 10 
2.4.  The Mre11:Rad50:Nbs1 (MRN) complex – a key player in DSB repair ......................... 11 
2.4.1.  Structural organization of the MR(N) complex........................................................ 12 
2.4.2.  Functional insights into thmplex ........................................................... 15 
2.4.3.  Multiple roles of MRN in eukaryotes ....................................................................... 17 
2.4.4.  Role of MR in prokaryotes ....................................................................................... 21 
2.4.5.  The bacterial MR catalytic head complex ................................................................ 22 
2.4.6.  Working model of MR´s DNA binding mechanism ................................................ 24 
2.5.  Objectives ......................................................................................................................... 26 
3.  Materials and Methods ............................................................................................................. 27 
3.1.  Materials ........................................................................................................................... 27 
3.1.1.  Oligonucleotides ....................................................................................................... 27 
3.1.2.  Plasmids ................................................................................................................... 29 
3.1.3.  Strains ....................................................................................................................... 31 
3.1.4.  Media and antibiotics ............................................................................................... 31 
3.1.5.  Preparation of sodium orthovanadate solution ......................................................... 31 
3.2.  Molecular biology methods .............................................................................................. 32 
3.2.1.  Molecular cloning .................................................................................................... 32 
3.2.2.  Site directed mutagenesis ......................................................................................... 33 
3.2.3.  Transformation of E. coli ......................................................................................... 34 
Table of Contents ii

3.3.  Protein biochemistry methods .......................................................................................... 34 
3.3.1.  Protein expression in E. coli ..................................................................................... 34 
3.3.2.  Purification of recombinant proteins ........................................................................ 34 
3.3.3.  Denaturing polyacrylamide gel electrophoresis (SDS-PAGE) ................................ 37 
HLH NBD3.3.4.  Dimerization of TmMre11 :Rad50 .................................................................. 37 
3.3.5.  Analytical size exclusion chromatography ............................................................... 37 
3.3.6.  Crosslinking via Bis-Maleimidoethane (BMOE) ..................................................... 38 
3.3.7.  Disulfide bridging via copper sulfate (CuSO ) ........................................................ 38 4
3.4.  Structural biology methods .............................................................................................. 39 
3.4.1.  Crystallization .......................................................................................................... 39 
3.4.2.  Data collection .......................................................................................................... 41 
3.4.3.  Structure determination, model building and refinement ......................................... 41 
3.4.4.  Small angle x-ray scattering ..................................................................................... 43 
3.5.  In vitro activity assays of Mre11:Rad50 .......................................................................... 43 
3.5.1.  ATP hydrolysis assay ............................................................................................... 43 
3.5.2.  Electrophoretic mobility shift assay (EMSA) .......................................................... 44 
3.5.3.  Nuclease activity assay ............................................................................................. 45 
3.5.4.  Endonuclease activity assay ..................................................................................... 45 
3.5.5.  Protein-DNA encircling assay .................................................................................. 46 
3.5.6.  Antibody DNA binding assay 46 
3.6.  Bioinformatic methods ..................................................................................................... 47 
3.6.1.  Structure based sequence alignment ......................................................................... 47 
4.  Results ........................................................