Analyzing protein-nucleic acid complexes using hybrid methods [Elektronische Ressource] : I. the DNA damage checkpoint protein DisA; II. structural biochemistry of RNA turnover by the exosome / Sophia Hartung

Sophia Hartung

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2008
• Nombre de lectures: 11
• 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
Analyzing Protein - Nucleic Acid Complexes
using Hybrid Methods
I. The DNA Damage Checkpoint Protein DisA
II. Structural Biochemistry of RNA Turnover by the Exosome
Sophia Hartung
aus
Würzburg
München, 2008Erklärung
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 Hilfe erarbeitet.
München, am 01.9.2008

....................................................
(Sophia Hartung)
Dissertation eingereicht am 01.09.2008
1. Gutachter Herr Prof. Dr. Karl-Peter Hopfner
2. Gutachter Herr Prof. Dr. Roland Beckmann
Mündliche Prüfung am 14. Oktober 2008
The presented thesis was prepared in the time from January 2005 to July 2008 in the laboratory
of Professor Dr. Karl-Peter Hopfner at the Gene Center of the Ludwig-Maximilians-Univers ity
of Munich (LMU).Parts of this PhD thesis have been published:
Hartung, S. and Hopfner K. P. (2007).
The exosome, plugged. EMBO Rep 8(5): 456-7.
* *
Witte, G., Hartung, S. , Büttner, K. and Hopfner K. P. (2008).
Structural biochemistry of a bacterial checkpoint protein reveals diadenylate cyclase activity
regulated by DNA recombination intermediates. Mol Cell 30(2): 167-78.
* These authors contributed equally to this work. Table of Contents
1 Analysis of Large Protein Complexes and Their Ligands .................................... 1 ........................
1.1 Preface...........................................................................................................................1 ........
1.2 Methods for Structural Analysis ...........................................................................................1 .
1.2.1 X-ray Crystallography......................................................................................2 .............
1.2.1.1 Prefa..........................................................................................ce 2 .........................
1.2.1.2 Crystallizing Proteins3 ..
1.2.1.3 Structure Determination by X-ray Diffraction ......................................................4 .
1.2.1.4 The Phase Probl........................................................................em 5 ........................
1.2.1.5 Refineme........................................................................................................nt 6 .....
1.2.2 SAX....................................................................................................................S 7 .........
1.2.2.1 Preface 7 .........................
1.2.2.2 Structure Determination by Small Angle X-ray Scattering ................ 8 ...................
1.2.2.3 The Guinier Approximation and Porod's Law..................................... 10 ................
1.2.2.4 The Pair Distribution Function................................................... 11 .........................
1.2.2.5 Ab initio Modeli..........................................................................................ng 12 .....
1.2.2.6 Computation of Scattering Patterns from Crystal Structures ...........13...................
1.3 Biochemical Activity Assay..............................................................................................s 14 .
2 Material and Methods .............................................................................................16 ....................
2.1 Materia..........................................................................................................l 16 .....................
2.2 Software......................................................................................................16 ........................
2.3 Methods .......................................................................................................................17 ........
2.3.1 Cloning, Expression and Purification Methods 17 .......
2.3.1.1 Cloni....................................................................................ng 17 ............................
2.3.1.2 Protein Expres................................................................................sion 18 ...............
2.3.1.3 Purification of Exosome Proteins and Complexes..................... 19 .........................
2.3.1.4 Purification of Exosome Complexes with Bound RNA ....................... 19 ...............
2.3.2 Biochemical Ass..............................................................................................ays 20 .......
2.3.2.1 Diadenylate Cyclase A..........................................................ssays 20 ......................
2.3.2.2 RNAse Ass.........................................................................ays 21 ............................
2.3.2.2.1 Radioactive labeling of oligonucleotides..........................................21 ...........
2.3.2.2.2 Assay Condi...........................................................tions 21 ..............................
2.3.2.2.3 Urea Gel Electrophore .........................................................sis 21 ....................
2.3.2.3 EMSA (electrophoretic mobility shift assa..........................y) 22 ............................
2.3.2.4 Fluorescent Labeling of the Exosome for Single-Molecule Experiment..s ..22.......
2.3.3 Crystallization and Structure Determination .................................................23 ..............
2.3.3.1 Exosome in Complex With Small RNA Molecules........................................23 .....
2.3.3.2 Csl4 S1-ZnR Dom..............................................................ain 23 ............................
2.3.3.3 Exosome core with copurified RN..................................................................A 24 ..
2.3.4 SAXS Experiments and Data Processing.......................................................24 .............
2.3.4.1 D..........................................................................................................isA 24 ...........
2.3.4.2 Exos..................................................................................ome 25 ............................
3 The DNA Integrity Scanning Protein A (DisA) ........................................................26 ..................
3.1 The DisA Protein and its Influence on Sporulation .............................................................26
3.2 The Crystal Structure of T. maritima DisA27 ..................
3.3 Cyclic Purine Nucleotides As Second Messengers .................................................29 ............
3.3.1 cAMP and cGMP ........................................................................................................29 .
3.3.2 C-di-GMP and Associated Enzymes................................................... 30 ........................
3.3.2.1 Diguanylate Cyclase Activity and GGDEF Domains.....................................31 .....
3.3.2.2 Phosphodiesterase Activity and EAL Doma...................................ins 32 ................3.3.2.3 PilZ as First Proposed c-di-GMP Binding Domain............................. 33 ................
3.4 The Role of Holliday Junctions and Fork Structures in DNA Repair ....................... 33 ..........
3.4.1 Double Strand Breaks............................................................................................34 ......
3.4.2 Stalled Replication Forks ..................................................................35 ..........................
3.4.3 DNA Damage Checkpoints 35 .......................
3.5 Aim Of The Project .........................................................................................35 ....................
4 Results – DisA .....................................................................................................37 .......................
4.1 Identification of the Correct Quaternary Structure of DisA ..............................................37 ..
4.2 Identification of the Enzymatic Activity of DisA ......................................... 41 ......................
4.2.1 DisA is a Specific Diadenylate Cyclase 41 ........................
4.2.2 The Influence of Mg2+ and Active Site Mutants on the Activity ...........44....................
4.3 The Influence of Different DNA Molecules on DisA Activity ................. 44..........................
4.4 The Influence of Azide ..............................................................................................46 ..........
4.5 DisA and DNA Binding ..........................................................................47 ............................
5 Discussion – DisA .........................................................................................49 .............................
5.1 SAXS as Complementary Method to X-ray Crystallography .....................................49 ........
5.2 The DisA Octamer............................................................................................................49 ...
5.3 Reliability of SAXS Structures .........................................................................50 ..................
5.4 The Moving Foci of DisA..................................................................................51 .................
5.5 c-di-AMP and Diadenylate Cyclase Activity ..............