A systematic investigation of DNA double strand break repair by a genome-wide RNAi screen in human cells:
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| Format: | Abschlussarbeit Buch |
| Sprache: | English |
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2008
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| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | XVI, 139 S. Ill., graph. Darst. |
Internformat
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| 100 | 1 | |a Słabicki, Mikołaj Michał |e Verfasser |4 aut | |
| 245 | 1 | 0 | |a A systematic investigation of DNA double strand break repair by a genome-wide RNAi screen in human cells |c presented by Mikołaj Michał Słabicki |
| 264 | 1 | |c 2008 | |
| 300 | |a XVI, 139 S. |b Ill., graph. Darst. | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 502 | |a Dresden, Techn. Univ., Diss., 2008 | ||
| 655 | 7 | |0 (DE-588)4113937-9 |a Hochschulschrift |2 gnd-content | |
| 856 | 4 | 2 | |m HBZ Datenaustausch |q application/pdf |u http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=022215646&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |3 Inhaltsverzeichnis |
| 999 | |a oai:aleph.bib-bvb.de:BVB01-022215646 | ||
Datensatz im Suchindex
| _version_ | 1804145276633481216 |
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| adam_text | Titel: A systematic investigation of DNA double strand break repair by a genome wide RNAi screen in human c
Autor: Słabicki, Mickołaj Michał
Jahr: 2008
Contents
1 Introduction 1
1.1 Effects of DNA damage 1
1.2 Chemical variety of sources of DNA damage 1
1.3 Consequences of DNA damage 2
1.4 Different DNA damage repair mechanisms 2
1.4.1 Direct repair 2
1.4.2 Nudeotide excision repair 2
1.4.3 Base excision repair 3
1.4.4 Mismatch repair 3
1.4.5 DNA double strand break repair 4
1.4.5.1 Response to DNA double strand break 4
1.4.5.2 Repair pathways 5
1.4.5.3 Nonhomologous end joining 5
1.4.5.4 Single strand annealing 5
1.4.5.5 Homologous recombination repair 6
1.5 DNA repair defects and its connection to diseases 7
1.6 DNA repair in cancer predisposition and development 8
1.7 Antitumor treatment - exploration of DNA repair pathways 9
1.8 Aim of the thesis 10
2 Results 11
2.1 Screening set up 11
2.1.1 EsiRNA: a loss of function tool for genome scale screens in mammalian cells 11
2.1.2 Assay for homologous recombination repair 13
2.1.3 Establishing a high throughput cell based assay for DSB repair 14
2.1.4 Performing the genome-scale screen 16
2.2 Results of the genome scale screen 17
2.2.1 Description of hits that decrease frequency of homologous recombination 21
2.2.2 Description of hits that increase frequency of homologous recombination 24
2.2.3 Description of hits that increase the intensity of the GFP signal 25
2.3 Characterization of selected hits 27
2.3.1 BAC tagging approach 27
2.3.2 Characterization of Rad51 and SHFM1 28
2.3.3 Characterization of K1AA0415 35
2.3.3.1 KIAA0415 is required for homologous recombination repair 35
2.3.3.2 Bioinformatics analysis of KIAA0415 36
2.3.3.3 KIAA0415 - in vivo rescue 41
2.3.3.3.1 In vivo rescue of the UV sensitive mutant 41
2.3.3.3.2 In vivo rescue of the chromosomal DNA DSB sensitive mutant 42
2.3.3.4 Purification of KIAA0415 44
2.3.3.5 In vitro binding to synthetic Holliday junction assay 47
2.3.3.6 Localization and immunoprecipitation of KIAA0415 50
2.3.4 Characterization of Clorf63 59
2.3.4.1 Clorf63 is required for homologous recombination repair 59
2.3.4.2 Clorf63 is a SR protein 60
2.3.4.3 Immunoprecipitation of mClorf63 61
2.3.4.4 mClorf63 localizes to RNA speckles 65
2.3.4.5 Subcellular localization of HUWE1 69
2.3.4.6 Direct interactors of Clorf63 70
XI
2.3.4.7 Increased drag sensitivity after silencing of Clorf63 72
2.3.5 Characterization of KIAA1604 (FLJ23325) • 73
2.3.5.1 KIAA1604 is required for homologous recombination repair 73
2.3.5.2 Localization of KIAA1604 74
2.3.5.3 Immunoprecipitation of KJAA1604 75
2.3.5.4 KIAA1604 and gamma-H2AX 76
3 Discussion 79
3.1 EsiRNA library resource 79
3.2 Set up of the screen 79
3.3 Performing the genome scale screen 80
3.4 Characterization of Rad51 and SHFM1 81
3.5 Characterization of KIAA0415 82
3.6 Characterization of Clorf63/Huwel 84
3.7 Characterization of KIAA1604 85
3.8 Summary of discussion 86
4 Materials and Methods 87
4.1 General buffers 87
4.1.1 Materials 87
4.1.2 Buffer recipes 88
4.1.3 Bacteria medium recipes 89
4.1.4 Yeast medium recipe 89
4.2 Cell Culture 90
4.2.1 Materials 90
4.2.2 Protocols 90
4.3 Preparation of esiRNA library 91
4.4 Performing the genome-scale screen 92
4.4.1 Materials 92
4.4.2 Methods 92
4.5 DNA cloning 94
4.5.1 Purification of plasmid DNA 94
4.5.2 Restriction enzyme digestion 94
4.5.3 DNA gel electrophoresis 94
4.5.4 PCR Z.ZZZZZZZ ZZZZ.Z Z 94
4.5.5 LigationofDNA 95
4.5.6 BAC engineering technology 95
4.6 esiRNA transfection 97
4.6.1 Forward transfection in 3.5 cm2 dish for mRN A isolation 97
4.6.2 Reverse transfection of esiRNA in 96 well format 97
4.6.3 Reverse transfection of esiRNA in 384 well format 97
4.7 Examination of gene knockdown on the mRNA level 98
4.7.1 Isolation of total RNA and cDNA synthesis 98
4.7.2 Quantitative PCR 98
4.8 Immunoprecipitation 99
4.8.1 Immunoprecipitation with anti-GFP antibody 99
4.8.1.1 Materials 99
4.8.1.2 Buffer recipes 99
4.8.1.3 Methods ZZZZZ^ZZZZZZZZ 1°2
4.8.1.3.1 Preparation of cell extract 102
XII
4.8.1.3.2 Coupling of anti-GFP antibody to G Sepharose beads 102
4.8.1.3.3 Immunoprecipitation protocol 103
4.8.2 HIS tag purification 103
4.8.2.1 Materials 103
4.8.2.2 Buffer recipes 104
4.8.2.3 Methods 104
4.8.2.3.1 Purification from bacteria 104
4.8.2.3.2 Purification after in vitro transcription and translation 105
4.8.2.3.3 Purification from HEK 293 105
4.8.3 Analysis of immunoprecipitation experiments 106
4.8.3.1 Running SDS-PAGE gels 106
4.8.3.2 Silver staining of SDS-PAGE gel 106
4.8.3.2.1 Materials 106
4.8.3.2.2 Buffer recipes 106
4.8.3.2.3 Methods 107
4.8.3.3 Western Blotting 107
4.8.3.3.1 Materials 107
4.8.3.3.2 Buffer recipes 108
4.8.3.3.3 Method 108
4.8.3.4 Mass spectrometry analysis 109
4.9 Imaging 110
4.9.1 Immunofluorescence staining 110
4.9.1.1 Materials 110
4.9.1.2 Buffer recipes 111
4.9.1.3 Methods Ill
4.9.1.3.1 Staining of cells on the cover slip 111
4.9.1.3.2 Staining of cells in 384 well plate 112
4.9.2 In vivo imaging 112
4.10 Rescue of UV and DNA chromosomal DSB sensitive bacteria mutants 113
4.10.1 Materials 113
4.10.2 Preparation of competent cells 113
4.10.3 Transformation of competent bacteria cells 113
4.10.4 In vivo rescue of the UV sensitive mutant 114
4.10.5 In vivo rescue of the chromosomal DNA double strand breaks 114
4.11 In vitro Holliday junction assay 115
4.11.1 Materials 115
4.11.2 Buffer recipes 115
4.11.3 Methods 116
4.11.3.1 Synthetic Holliday junction preparation 116
4.11.3.2 Performing binding assay 116
4.12 Yeast-two-Hybrid (Y2H) 117
4.12.1 Materials 117
4.12.2 Buffer recipes 117
4.12.3 System description 118
4.12.4 Preparation of yeast competent cells 118
4.12.5 Plasmid linearization 119
4.12.6 PCR amplification of the gene fragments 119
4.12.7 GAP repair 119
4.12.8 Mating of yeast strains 120
4.12.9 X-gal assay 120
5 Supplementary Materials 121
6 References 127
7 Acknowledgments 137
8 Declaration 139
XIII
List of Figures
Figure 1. Mechanism of double strand break repair 7
Figure 2. Flow chart of the esiRNA library preparation 12
Figure 3. Schematic representation of the DR-GFP assay used in the genome scale screen ... 14
Figure 4. Layout of DR-GFP genome scale screen and respond of control genes 16
Figure 5. Results of the genome scale screen and validation of the hits that decreased and
increased the frequency of homologous recombination 19
Figure 6. Results of the genome scale screen and validation of the hits that increased intensity
oftheGFP signal 20
Figure 7. FACS flow histogram of a GFP intensity hit 25
Figure 8. Localization study of mRad51-LAP and mSHFMl 29
Figure 9. Purification of mRad51-LAP and mSHFMl-LAP 30
Figure 10. Trypsin cleavage sites in the SHFM1 sequence 31
Figure 11. DR-GFP phenotype, mRNA levels and viability after knockdown of KIAA0415...35
Figure 12. Phylogenetic tree of multiple alignment of human KIAA0415 36
Figure 13. Stretch of homology between human KIAA0415 and E. colt RecO 37
Figure 14. Threading of K1AA0415 38
Figure 15. Sequence alignment of wedge domain from bacterial RecG and KIAA0415 39
Figure 16. Sequence comparison of TRG motif 40
Figure 17. In vivo rescue experiment of UV sensitive mutant strain bacteria 42
Figure 18. In vivo rescue experiment of chromosomal DNA DSB sensitivity assay 43
Figure 19. Purification strategies of KIAA0415 46
Figure 20. Holliday junction binding experiment 48
Figure 21. Purification of KIAA0415 from He La cells 51
Figure 22. Localization of tagged KIAA0415 52
Figure 23. Reduction of homologous recombination repair after C20orf29 knockdown 53
Figure 24. Analysis of SPG11-LAP expressed in HeLa cells 56
Figure 25. DR-GFP phenotype, mRNA levels and viability after knockdown of Clorf63 59
Figure 26. Analysis of the Clorf63 sequence 61
Figure 27. Analysis of mClorf63-LAP in HeLa cells 62
Figure 28. Immunofluorescence of mClorf63-LAP 65
Figure 29. Colocalization of mClorf63-LAP with different repair foci 65
Figure 30. Colocalization of mClorf63-LAP with a speckles marker (SM) 66
Figure 31. Localization of mClorf63-LAP during cell cycle 67
Figure 32. Formation of the mClorf63 -LAP speckle 68
Figure 33. Effect of different triggers on mClorf63-LAP localization 68
Figure 34. Localization of Huwel in response to DNA damaging agent 70
Figure 35. X-gal assay of interaction between Clorf63 and putative interactors 71
Figure 36. Sensitivity towards different drugs after the Clorf63 knockdown 72
Figure 37. DR-GFP phenotype and viability after knockdown of KIAA1604 73
Figure 38. Analysis of mKIAA1604-LAP 74
Figure 39. Quantification of gamma-H2AX foci after KIAA1604 knockdown 77
XIV
List of Tables
Table 1. Mass spectrometry results of mRad51-LAP and mSHFMl-LAP purification 33
Table 2. Mass spectrometry results of K1AA0415 purification 54
Table 3. Mass spectrometry result of SPG11-LAP purification 56
Table 4. Mass spectrometry results of mClorf63-LAP purification 62
Table 5. Mass spectrometry results of mKIAA1604-LAP purification 75
Table 6. Genes tagged by BAC tagging engineering technology 96
Table 7. Primer sequences used for Q-PCR 98
Table 8. Primer sequences used for synthetic Holliday junction 115
List of Supplementary Tables
Supplementary Table 1. Genes that after knockdown reproducible decreased frequency of
homologous recombination with two independent esiRNAs 121
Supplementary Table 2. Genes that after knockdown reproducible increase the frequency of
homologous recombination with two independent esiRNAs 122
Supplementary Table 3. Genes that after knockdown reproducible increase the intensity of the
GFP signal with two independent esiRNAs 123
Supplementary Table 4. Primer sequences for two independent esiRNA targeting verified hits
from the screen that decreased frequency of homologous
recombination 124
Supplementary Table 5. Primer sequences for two independent esiRNA targeting verified hits
from the screen that increased frequency of homologous
recombination 125
Supplementary Table 6. Primer sequences for two independent esiRNA targeting verified hits
from the screen that increased intensity of the GFP signal 126
XV
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| any_adam_object | 1 |
| author | Słabicki, Mikołaj Michał |
| author_facet | Słabicki, Mikołaj Michał |
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| author_sort | Słabicki, Mikołaj Michał |
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| bvnumber | BV026668963 |
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| indexdate | 2024-07-09T23:16:53Z |
| institution | BVB |
| language | English |
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| physical | XVI, 139 S. Ill., graph. Darst. |
| publishDate | 2008 |
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| spelling | Słabicki, Mikołaj Michał Verfasser aut A systematic investigation of DNA double strand break repair by a genome-wide RNAi screen in human cells presented by Mikołaj Michał Słabicki 2008 XVI, 139 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Dresden, Techn. Univ., Diss., 2008 (DE-588)4113937-9 Hochschulschrift gnd-content HBZ Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=022215646&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Słabicki, Mikołaj Michał A systematic investigation of DNA double strand break repair by a genome-wide RNAi screen in human cells |
| subject_GND | (DE-588)4113937-9 |
| title | A systematic investigation of DNA double strand break repair by a genome-wide RNAi screen in human cells |
| title_auth | A systematic investigation of DNA double strand break repair by a genome-wide RNAi screen in human cells |
| title_exact_search | A systematic investigation of DNA double strand break repair by a genome-wide RNAi screen in human cells |
| title_full | A systematic investigation of DNA double strand break repair by a genome-wide RNAi screen in human cells presented by Mikołaj Michał Słabicki |
| title_fullStr | A systematic investigation of DNA double strand break repair by a genome-wide RNAi screen in human cells presented by Mikołaj Michał Słabicki |
| title_full_unstemmed | A systematic investigation of DNA double strand break repair by a genome-wide RNAi screen in human cells presented by Mikołaj Michał Słabicki |
| title_short | A systematic investigation of DNA double strand break repair by a genome-wide RNAi screen in human cells |
| title_sort | a systematic investigation of dna double strand break repair by a genome wide rnai screen in human cells |
| topic_facet | Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=022215646&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT słabickimikołajmichał asystematicinvestigationofdnadoublestrandbreakrepairbyagenomewidernaiscreeninhumancells |