Structural and functional characterization of nucleotide excision repair proteins:
Gespeichert in:
| 1. Verfasser: | |
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| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
2011
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| Schlagworte: | |
| Online-Zugang: | Volltext Inhaltsverzeichnis |
| Beschreibung: | Zsfassung in dt. und engl. Sprache. - Online erschienen: 2013 |
| Beschreibung: | 116 Bl. Ill., graph. Darst. |
Internformat
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| 100 | 1 | |a Wolski, Stefanie Carola |e Verfasser |0 (DE-588)1031288686 |4 aut | |
| 245 | 1 | 0 | |a Structural and functional characterization of nucleotide excision repair proteins |c von Stefanie Carola Wolski |
| 246 | 1 | 3 | |a Strukturelle und funktionelle Charakterisierung von Nucleotid-Exzisions-Reparatur Proteinen |
| 264 | 1 | |c 2011 | |
| 300 | |a 116 Bl. |b Ill., graph. Darst. | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
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Datensatz im Suchindex
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|---|---|
| adam_text | IMAGE 1
TABLE OF CONTENTS
SUMMARY 1
ZUSAMMENFASSUNG 2
1 INTRODUCTION 4
1.1 LESIONS ADDRESSED BY NUCLEOTIDE EXCISION REPAIR 4
1.2 MOLECULAR MECHANISM OF EUKARYOTIC NER 6
1.3 INSIGHTS INTO THE MECHANISM AND FUNCTIONS OF THE XPD PROTEIN 10
1.4 DISEASES CAUSED BY MUTATIONS IN XPD 12
1.5 IRON-SULFUR CLUSTERS 14
1.6 ARCHAEAL HOMOLOGS: A MODEL FOR EUKARYOTIC NER 15
2 MATERIALS 18
2.1 CHEMICALS 18
2.2 EQUIPMENT, INSTRUMENTATION AND EXPENDABLE MATERIALS 18
2.3 BACTERIAL CELL CULTURE 20
2.3.1 BACTERIAL STRAINS 20
2.3.2 EXPRESSION PLASMIDS 20
2.3.3 MEDIA FOR BACTERIAL CELL CULTURE 20
2.3.4 MEDIA ADDITIVES 2 1
2.4 ENZYMES 21
2.5 NUCLEOTIDES AND OLIGONUCLEOTIDES 21
2.6 BUFFER AND SOLUTIONS 23
2.7 OXIDANTS AND REDUCING AGENTS 24
2.8 KITS 24
2.9 CRYSTALLIZATION SCREENS 24
2.10 COMPUTER SOFTWARE 25
3 METHODS 26
3.1 MOLECULAR BIOLOGY TECHNIQUES 26
3.1.1 MUTAGENESIS 26
3.1.2 PLASMID ISOLATION 27
3.1.3 LABELING AND DUPLEXING OF SINGLE-STRANDED DNA 27
3.1.4 TRANSFORMATION 27
3.2. PROTEIN EXPRESSION 28
3.3 PROTEIN PURIFICATION 28
3.3.1 CELL LYSIS 28
3.3.2 AFFINITY CHROMATOGRAPHY - NI 2+ COLUMN 29
HTTP://D-NB.INFO/1032228784
IMAGE 2
3.3.3 SIZE EXCLUSION CHROMATOGRAPHY 29
3.4 PROTEIN CHARACTERIZATION 30
3.4.1 SDS-PAGE ANALYSIS 30
3.4.2 DETERMINATION OF THE PROTEIN CONCENTRATION 30
3.4.3 CIRCULAR DICHROISM SPECTROSCOPY 3 1
3.4.4 THERMOFLUOR ANALYSIS 3 1
3.4.5 IN VITRO ATPASE ASSAY 32
3.4.6 BIOLAYER INTERFEROMETRY 33
3.4.7 GEL-BASED HELICASE ASSAY 33
3.4.8. FLUORESCENCE BASED HELICASE ASSAY 34
3.4.9 ELECTRON PARAMAGNETIC RESONANCE (EPR) 34
3.5 CRYSTALLIZATION 36
3.6 X-RAY CRYSTALLOGRAPHY 37
3.6.1 DATA COLLECTION 37
3.6.2 MULTIWAVELENGTH ANOMALOUS DISPERSION (MAD) 39
3.6.3 MOLECULAR REPLACEMENT 40
3.6.4 MODEL BUILDING AND REFINEMENT 4 1
4 RESULTS 44
4.1 PROTEIN EXPRESSION AND PURIFICATION 44
4.1.1 PROTEIN EXPRESSION AND PURIFICATION OF XPD WT AND VARIANTS FROM
THERMOPLASMA
ACIDOPHILUM 44
4.1.2 PROTEIN EXPRESSION AND PURIFICATION OF XPD FROM FERROPLASMA
ACIDARMANUS 46
4.2 CD SPECTROSCOPY REVEALS THAT TAXPD AND FAXPD ARE PROPERLY FOLDED 47
4.3 CRYSTALLIZATION EXPERIMENTS 48
4.3.1 CRYSTALLIZATION OF TAXPD 48
4.3.2 CRYSTALLIZATION OF TAXPD WITH DNA 49
4.3.3 CRYSTALLIZATION OF FAXPD 50
4.4 TAXPD APO STRUCTURE 5 1
4.4.1 DATA COLLECTION AND STRUCTURE SOLUTION OF APO TAXPD 5 1
4.4.2 OVERALL TAXPD STRUCTURE 52
4.5 MODEL O F FAXPD BASED ON TAXPD 55
4.6 XPD-DNA BINDING MODEL 56
4.7 TAXPD-DNA COMPLEX 59
4.7.1 DATA COLLECTION AND STRUCTURE SOLUTION OF TAXPD WITH DNA 59
4.7.2 XPD-DNA STRUCTURE 60
IMAGE 3
4.8 SEQUENCE ALIGNMENT OF XPD FROM DIFFERENT ORGANISMS 61
4.9 SITE-DIRECTED MUTAGENESIS 62
4.10 THERMOFLUOR OF TAXPD MUTANTS 63
4.11 BIOLAYER INTERFEROMETRY IDENTIFIES RESIDUES WHICH ARE INVOLVED IN
DNA BINDING 64
4.12 TAXPDS ATPASE ACTIVITY IS STIMULATED BY SSDNA 66
4.13 TAXPD IS AN ATP-DEPENDENT 5 -3 HELICASE AND RESIDUES Y166, D582 AND
R584 ARE IMPORTANT
FOR HELICASE ACTIVITY 68
4.14 TAXPD AND FAXPD BIND FLUORESCEIN-CONTAINING BUBBLE SUBSTRATES WITH
A HIGHER AFFINITY AS
NON-DAMAGED BUBBLE SUBSTRATES 69
4.15 IRON SULFUR CLUSTER ANALYSIS 7 1
4.15.1 ELECTRON PARAMAGNETIC RESONANCE STUDIES 7 1
4.15.2 IRON-SULFUR CLUSTER ANALYSIS OF TAXPD VARIANTS 73
4.15.3 TAXPD WT SHOWS REDUCED HELICASE ACTIVITY IN THE PRESENCE OF
OXIDIZING AND REDUCING
AGENTS 75
4.15.4 XPD IS FOLDED PROPERLY IN THE PRESENCE OF DIFFERENT OXIDANTS AND
REDUCING AGENTS 76
5 DISCUSSION 77
5.1 THE XPD CRYSTAL STRUCTURE 77
5.2 DISEASE CAUSING MUTATIONS 79
5.3 CONCLUSIONS FROM THE MUTAGENESIS STUDIES 83
5.4 THE ROLE OF THE FES CLUSTER 85
5.5 MECHANISTIC CONCLUSIONS FROM THE XPD STRUCTURE 89
5.7 FINAL CONCLUSIONS 9 1
6 REFERENCES 92
7 APENDIX 104
7.1 ABREVIATIONS 104
7.2 TABLE OF TABLE AND FIGURES 106
7.3 SUPERPOSITION OF XPD WITH UVRB, NS3 AND HEL308 107
7.4 SEQUENCES OF CONSTRUCTS 108
7.5 PRIMER FOR MUTAGENESIS I L L
ACKNOWLEDGEMENTS 114
AFFIDAVIT 115
LIST OF PUBLICATIONS 116
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| spelling | Wolski, Stefanie Carola Verfasser (DE-588)1031288686 aut Structural and functional characterization of nucleotide excision repair proteins von Stefanie Carola Wolski Strukturelle und funktionelle Charakterisierung von Nucleotid-Exzisions-Reparatur Proteinen 2011 116 Bl. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Zsfassung in dt. und engl. Sprache. - Online erschienen: 2013 Würzburg, Univ., Diss., 2011 DNS-Reparatur (DE-588)4150347-8 gnd rswk-swf Helicasen (DE-588)4159496-4 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content DNS-Reparatur (DE-588)4150347-8 s Helicasen (DE-588)4159496-4 s DE-604 Erscheint auch als Online-Ausgabe urn:nbn:de:bvb:20-opus-67183 https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-67183 Resolving-System kostenfrei Volltext DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=025728352&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Wolski, Stefanie Carola Structural and functional characterization of nucleotide excision repair proteins DNS-Reparatur (DE-588)4150347-8 gnd Helicasen (DE-588)4159496-4 gnd |
| subject_GND | (DE-588)4150347-8 (DE-588)4159496-4 (DE-588)4113937-9 |
| title | Structural and functional characterization of nucleotide excision repair proteins |
| title_alt | Strukturelle und funktionelle Charakterisierung von Nucleotid-Exzisions-Reparatur Proteinen |
| title_auth | Structural and functional characterization of nucleotide excision repair proteins |
| title_exact_search | Structural and functional characterization of nucleotide excision repair proteins |
| title_full | Structural and functional characterization of nucleotide excision repair proteins von Stefanie Carola Wolski |
| title_fullStr | Structural and functional characterization of nucleotide excision repair proteins von Stefanie Carola Wolski |
| title_full_unstemmed | Structural and functional characterization of nucleotide excision repair proteins von Stefanie Carola Wolski |
| title_short | Structural and functional characterization of nucleotide excision repair proteins |
| title_sort | structural and functional characterization of nucleotide excision repair proteins |
| topic | DNS-Reparatur (DE-588)4150347-8 gnd Helicasen (DE-588)4159496-4 gnd |
| topic_facet | DNS-Reparatur Helicasen Hochschulschrift |
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