Early events in DNA double strand break repair after damage induction with a laser microbeam [Elektronische Ressource] / von Paulius Grigaravičius

Dekanat

Sujets

• Biologie

Informations

• Publié par: friedrich-schiller-universitat_jena
• Publié le: 01 janvier 2009
• Nombre de lectures: 46
• Langue: English
• Poids de l'ouvrage: 13 Mo

Extrait

Early events in DNA double strand break repair after
damage induction with a laser microbeam





Dissertation
zur Erlangung des akademischen Grades
doctor rerum naturalium (Dr. rer. nat.)




vorgelegt dem Rat der Biologisch-Pharmazeutischen Fakultät
der Friedrich-Schiller- Universität Jena



von Dipl. Biophys.

Paulius Grigaravi čius

geboren am 30. März 1979
in Vilnius, Litauen
































This work was performed in




Supervisor: Prof. Dr. Karl Otto Greulich
Leibniz Institute for Age Research – Fritz Lipmann Institute

Gutachter: Prof. Dr. Zhao Qi Wang

Gutachter: Prof. Dr. Cristina Cardoso
Molecular Cell Biology
Technical University Darmstadt



Day of defense: 18.06.09
Content

Content
Content................................................................................................................................................I
Summary..................III
Zusammenfassung...........................................................................................................................IV
1. Introduction................................................................................................................................... 1
1.1. DNA double strand break repair ............................................................................................... 2
1.2. Laser-microbeam as a precise tool to study DNA damage and repair ...................................... 6
1.3. Comet-assay for detection of DNA fragmentation ................................................................... 8
1.4. Aim of the thesis ..................................................................................................................... 10
2. Materials and Methods............................................................................................................... 12
2.1. Cell culture.............................................................................................................................. 12
2.2. Cloning.................................................................................................................................... 12
2.3. Transient transfection.............................................................................................................. 17
2.4. Stable cell line generation ....................................................................................................... 18
2.5. Western Blot analysis.............................................................................................................. 18
2.6. Immunohistochemistry............................................................................................................ 20
2.7. Immunofluorescent Comet-assay (IFCA) ............................................................................... 21
2.8. Damage induction and quantitative calculation of used photons, doses and molecule
numberby ionizing radiation, UV-A laser-microbeam irradiation and bleomycin treatment . 25
2.9. DNA damaging by laser irradiation ........................................................................................ 27
2.9.1. Optical setup for irradiation at 420-455 nm .................................................................... 27
2.9.2. Optical setup for irradiation at 350 nm during live cell imaging .................................... 28
2.10. Microscopy and image analysis ............................................................................................ 29
2.10.1. Live cell imaging ............................................................................................................ 29
2.10.2. Immunofluorescence....................................................................................................... 31
3. Results.......................................................................................................................................... 32
3.1. Laser induce damage with high spatial resolution .................................................................. 32
3.2. DNA damage dependency on irradiation quality.................................................................... 33
3.2.1. Different levels of DNA damage detected by γ-H2AX staining ........................................ 33
3.2.2. Pulse energy and dose dependent DNA damage induction.............................................. 35
3.2.3. Wavelength dependent DNA damage induction ............................................................... 36
3.3. Immunofluorescent Comet-assay – a novel staining method of CA for comparison of DNA
fragmentation levels in single cells......................................................................................... 37
3.3.1. IFCA versus conventional SYBR Green staining.............................................................. 37
3.3.2. SSBs in neutral Comet-assay and fragment size estimation............................................. 40
IContent

3.3.3. DNA fragmentation after ionizing radiation, UV-A laser-microbeam and chemical
damage ............................................................................................................................. 43
3.4. Timing of DNA repair proteins on laser induced DSBs ......................................................... 46
3.4.1. Generation of expression constructs and U-2 OS cell lines stably expressing fusion
proteins............................................................................................................................. 46
3.4.2. Recruitment of DSB repair proteins highly depend on the used laser pulse energy ........ 50
3.4.3. Late NHEJ factor XRCC4 is recruited earlier than NBS1 from the MRN complex ......... 53
3.4.4. XRCC4 recruitment time does not depend on the NBS1................................................... 56
3.4.5. XRCC4 is replaced by Rad51 ........................................................................................... 58
3.4.6. DNA-PKcs is phosphorylated after NHEJ and before HRR are recruited....................... 60
3.5. Spatial dynamics of laser induced damage ............................................................................. 61
3.5.1. Quasi continuous laser irradiation induce different levels of DSBs on laser track ......... 61
3.5.2. Laser induced foci fuse ~ 20 minutes after irradiation .................................................... 62
4. Discussion .................................................................................................................................... 64
4.1. DNA double strand break induction depends on a subtle combination of pulse peak power,
dose and wavelength of the laser-microbeam ......................................................................... 64
4.2. Fusion of laser induced damage.............................................................................................. 65
4.3. IFCA for high resolution visualisation of DNA fragmentation .............................................. 65
4.4. Fragment size calibration and direct comparison of fragmentation patterns after different
damaging treatments ............................................................................................................... 67
4.5. Inverse relationship of DSB repair protein recruitment time with pulse energy..................... 69
4.6. Early NHEJ is followed by recruitment of the MRN complex, DNA-PKcs
autophosphorylation and late HRR ......................................................................................... 70
4.7. Final conclusions/theses.......................................................................................................... 75
5. References.................................................................................................................................... 77
6. Appendix..................................................................................................................................... A1
6.1. Abbreviations......................................................................................................................... A1
6.2. Buffers and solutions ............................................................................................................. A4
6.3. Materials and Manufacturers.................................................................................................. A8
6.4. Sequencing........................................................................................................................... A15
Previous work on the protein-substrate interaction ................................................................. A19
CURRICULUM VITAE.............................................................................................................. A22
Eigenständigkeitserklärung.... A26
Acknowledgments......................................................................................................................... A27


IISummary

Summary