Dynamics of DNA repair factors and chromosomes studied by laser-UVA-microirradiation and laser photobleaching [Elektronische Ressource] / vorgelegt von Joachim Walter

ludwig-maximilians-universitat_munchen - Walter Hawkins , Christian Joachim

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Dynamics of DNA-repair factors and
chromosomes studied by laser-UVA-
microirradiation and laser-photobleaching

Joachim Walter

München 2003

Dynamics of DNA-repair factors and
chromosomes studied by laser-UVA-
microirradiation and laser-photobleaching

Joachim Walter

DISSERTATION
an der FAKULTÄT FÜR BIOLOGIE
LUDWIG-MAXIMILIANS-UNIVERSITÄT MÜNCHEN

vorgelegt von
Joachim Walter
aus Kassel

München, den 2. Juni 2003

Dissertation eingereicht am 2. Juni 2003

1. Gutachter: Prof. Dr. T. Cremer
2. Gutachter: Prof. Dr. F. Eckardt-Schupp

Tag der mündlichen Prüfung: 16. Oktober 2003

Destruction is not negative
you must destroy to build

from: „Zeichnungen des
Patienten OT“
Einstürzende Neubauten
Contents v

Contents
LIST OF FIGURES.................................................................................................. VIII
SUMMARY................................................................................................................. 1
1 INTRODUCTION ............................................................................................. 3
1.1 DNA double-strand break repair................................................................... 3
1.1.1 Phosphorylation of H2AX marks broken chromatin ...................................... 4
1.1.2 The Mre11-Rad50 complex .......................................................................... 5
1.1.3 Rad51........................................................................................................... 6
1.2 Laser-UV-microirradiation............................................................................. 6
1.3 Nuclear architecture and dynamics.............................................................. 6
1.3.1 The arrangement of chromosome territories in interphase cell nuclei .......... 7
1.3.2 The dynamics of nuclear proteins................................................................. 9
1.4 Confocal microscopy................................................................................... 10
1.5 Questions addressed in this work.............................................................. 12
2 MATERIALS AND PROTOCOLS.................................................................. 14
2.1 Cell culture ................................................................................................... 14
2.1.1 Cell types and culture media ...................................................................... 14
2.1.2 Starting a cell culture from frozen cells....................................................... 15
2.1.3 Subcultivation of adherently growing cells.................................................. 16
2.1.4 Seeding cells to coverslips at a defined ratio.............................................. 17
2.1.5 Synchronization of HeLa cells with aphidicolin ........................................... 18
2.1.6 Lipofection of GFP-Rad51 plasmid and NLS-vimentin with Pfx-1 or
Fugene 6 .................................................................................................... 18
2.2 Laser-UVA-microirradiation ........................................................................ 20
2.2.1 Preparation of gridded coverslips ............................................................... 20
2.2.2 Polylysine coating of coverslips.................................................................. 21
2.2.3 Preparation of cells for Laser-UVA-microirradiation.................................... 22
2.2.4 Laser-UVA-microirradiation with the P.A.L.M. microdissection system ...... 23
2.2.5 Laser-UVA-microirradiation at an LSM 510 with UV-laser.......................... 24
2.2.6 Fixation and immunofluorescence staining of Rad51 and Mre11 after
microirradiation........................................................................................... 25
2.2.7 Immunofluorescence staining of BrdU........................................................ 27
2.2.8 Antibodies used in this work ....................................................................... 29
2.2.9 Staining of nuclear DNA with Propidium Iodide (PI) ................................... 30
2.3 Microscopy................................................................................................... 30
2.3.1 Transmitted light microscopy...................................................................... 30
2.3.2 Epifluorescence microscopy with widefield excitation................................. 31
2.3.3 Laser-scanning microscopy........................................................................ 32
vi Contents

2.3.4 Measurement of the chromatic shift............................................................ 34
2.3.5 Preparation of cells for live cell microscopy................................................ 35
2.3.6 Laser-photo-bleaching and live cell microscopy at the LSM 410................ 37
2.4 Evaluation..................................................................................................... 38
2.4.1 Software 38
2.4.2 The fourfold-point correlation...................................................................... 39
3 RESULTS ...................................................................................................... 40
3.1 Localization and dynamics of DNA-repair proteins studied by laser-
UVA-microirradiation................................................................................... 40
3.1.1 Setup of the microirradiation systems......................................................... 40
3.1.1.1 The P.A.L.M. microdissection system......................................... 40
3.1.1.2 Adaption of a Zeiss LSM 510 for microirradiation....................... 41
3.1.2 Rad51 accumulates at irradiated nuclear sites in human fibroblasts.......... 44
3.1.3 Rad51 accumulation at irradiated sites is a specific reaction to DNA
damage 49
3.1.4 Mre11 accumulates at laser-UVA-microirradiated nuclear sites and
colocalizes with Rad51 accumulations ....................................................... 51
3.1.5 Accumulation of other DNA-repair related proteins after laser-UVA-
microirradiation........................................................................................... 55
3.1.6 The nuclear distribution of PML after microirradiation ................................ 56
3.1.7 The fraction of cells with focal Rad51 nuclear staining remains
unchanged after microirradiation with 30 nJ ............................................... 57
3.1.8 Chromosome territory arrangements during DSB repair ............................ 59
3.1.9 Localization of Rad51 in unirradiated nuclei 60
3.1.9.1 Endogenous Rad51 foci ............................................................. 60
3.1.9.2 GFP-Rad51 filaments................................................................. 62
3.2 Chromosome territory dynamics studied in living cells by
photobleaching of GFP-tagged chromatin (H2B-GFP) ............................. 65
3.2.1 Setup of a 4D live cell imaging system at an LSM 410............................... 65
3.2.2 Calibration of the detector response........................................................... 67
3.2.3 Stability of CT arrangements in interphase nuclei visualized by stripes of
photobleached H2B-GFP ........................................................................... 70
3.2.4 Changes of CT arrangements in mitosis visualized by unbleached H2B-
GFP tagged chromatin ............................................................................... 72
3.2.5 Quantitative evaluation of changes in the radial distribution of unbleached
chromatin in mitosis.................................................................................... 78
3.2.6 Quantitative evaluation of the degree of clustering of unbleached
chromatin by an autocorrelation method .................................................... 81
4 DISCUSSION................................................................................................. 87
4.1 Technical advances in laser-microirradiation ........................................... 87
4.2 Localization and dynamics of DNA-repair proteins .................................. 91
4.2.1 Rad51-accumulation at irradiated sites and its relation to Mre11 ............... 91
4.2.2 Persistence of Rad51 S-Phas