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 Nucleotide 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 ВАС tagging approach ............................ 27
2.3.2 Characterization of Rad51 and SHFM1 ............. 28
2.3.3 Characterization of KIAA0415 .................... 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
2.3.4.7 Increased drug 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 KIAA1604 ........ 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 ............................................. 94
4.5.5 Ligation of DNA ................................. 95
4.5.6 ВАС engineering technology ...................... 95
4.6 esiRNA transfection .................................... 97
4.6.1 Forward transfection in 3.5 cm2 dish for mRNA
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 ............................... 102
4.8.1.3.1 Preparation of cell
extract .................... 102
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 ............................... 111
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
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