Characterization of the SCFFbxo28 ubiquitin ligase in mitosis and in topoisomerase II alpha-dependent DNA decatenation:
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| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
Heidelberg
2015
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| Online-Zugang: | Inhaltsverzeichnis Inhaltsverzeichnis |
| Beschreibung: | Im Titel ist "Fbxo28" hochgestellt. |
| Beschreibung: | III, 116 Seiten Illustrationen, Diagramme 30 cm |
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| 100 | 1 | |a Schmitt, Miriam |e Verfasser |0 (DE-588)1130426238 |4 aut | |
| 245 | 1 | 0 | |a Characterization of the SCFFbxo28 ubiquitin ligase in mitosis and in topoisomerase II alpha-dependent DNA decatenation |c presented by Miriam Schmitt |
| 264 | 1 | |a Heidelberg |c 2015 | |
| 300 | |a III, 116 Seiten |b Illustrationen, Diagramme |c 30 cm | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 500 | |a Im Titel ist "Fbxo28" hochgestellt. | ||
| 502 | |b Dissertation |c Ruperto-Carola University of Heidelberg |d 2016 | ||
| 655 | 7 | |0 (DE-588)4113937-9 |a Hochschulschrift |2 gnd-content | |
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Datensatz im Suchindex
| _version_ | 1804179109672124416 |
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| adam_text | CHARACTERIZATION OF THE SCF
FBXO28
UBIQUITIN LIGASE IN
MITOSIS AND IN TOPOISOMERASE II ALPHA-DEPENDENT
DNA DECATENATION
DISSERTATION
SUBMITTED TO THE
COMBINED FACULTIES FOR THE NATURAL SCIENCES AND FOR MATHEMATICS
OF THE RUPERTO-CAROLA UNIVERSITY OF HEIDELBERG, GERMANY
FOR THE DEGREE OF
DOCTOR OF NATURAL SCIENCES
PRESENTED BY
MIRIAM SCHMITT
BORN IN: MANNHEIM-NECKARAU, GERMANY
2015
TABLE OF CONTENTS
SUMMARY.......................................................................................................................................I
ZUSAMMENFASSUNG....................................................................................................................
II
1
INTRODUCTION..............................................................................................................................1
1.1 CHROMOSOME INSTABILITY AND
CANCER..................................................................................
1
1.2 THE UBIQUITIN PROTEASOME SYSTEM (UPS)
.........................................................................
3
1.3
SCF-E3-LIGASES..................................................................................................................6
1.3.1 THE F-BOX
FAMILY.......................................................................................................
6
1.3.1.1
FBXO28.................................................................................................................7
1.4 BIOLOGICAL ROLES OF F-BOX PROTEINS IN CANCER
.....................................................................
9
1.5 ATYPICAL FUNCTIONS OF F-BOX
PROTEINS................................................................................11
1.6 DNA
TOPOISOMERASES......................................................................................................13
1.6.1 TYPE IIA
TOPOISOMERASES.......................................................................................
13
1.6.1.1 STRUCTURE AND CATALYTIC MECHANISM OF TYPE IIA ENZYMES
...............................
14
1.6.1.2 TOPO LLA REGULATION AND POST-TRANSLATIONAL MODIFICATION
...................................
15
1.6.1.3 ROLE OF TOPO LLA IN MITOSIS AND CHROMOSOME INSTABILITY
.................................
17
1.7
OBJECTIVES........................................................................................................................
18
2 MATERIAL AND
METHODS...........................................................................................................19
2.1 MATERIAL
OVERVIEW.............................................................................................................19
2.1.1 CHEMICALS AND REAGENTS
.....................................................
19
2.1.2 LABORATORY
EQUIPMENT.............................................................................................
22
2.1.3 BUFFERS AND
MEDIA...................................................................................................
24
2.1.4
ANTIBODIES.............................................................................................................
28
2.1.5
OLIGONUCLEOTIDES.....................................................................................................
29
2.1.6 DNA
VECTORS............................................................................................................
30
2.1.7 SMALL INTERFERING
RNAS............................................................................................31
2.1.8 BACTERIAL
STRAINS......................................................................................................
32
2.1.9 HUMAN CELL
LINES......................................................................................................
33
2.1.10 ANTIBIOTICS..................................... 34
2.1.11 PURCHASED
KITS......................................................................................................
34
2.2 METHODS
OVERVIEW...........................................................................................................
35
2.2.1 METHODS IN MOLECULAR CELL
BIOLOGY.........................................................................35
2.2.1.1 MAMMALIAN CELL
CULTURE....................................................................................
35
2.2.1.2 TRANSFECTION OF MAMMALIAN
CELLS......................................................................35
2.2.1.3 GENERATION OF STABLE CELL
LINES..........................................................................36
2.2.1.4 HARVESTING AND FREEZING MAMMALIAN CELLS
.......................................................
37
2.2.1.5 MG132 TREATMENT OF
CELLS.................................................................................
37
2.2.1.6 CELL CYCLE SYNCHRONISATION OF
CELLS....................................................................37
2.2.1.7 LIVE CELL
IMAGING................................................................................................37
2.2.1.8 IMMUNOFLUORESCENCE ANALYSIS AND COVERSLIP PREPARATION
...............................
38
2.2.2 METHODS IN PROTEIN
BIOCHEMISTRY............................................................................39
2.2.2.1 PROTEIN EXTRACT PREPARATION OF
CELLS...................................................................39
2.2.2.2 DETERMINATION OF PROTEIN CONCENTRATION
...........................................................
39
2.2.2 3 SDS-PAGE AND WESTERN
BLOT.........................................................................39
2.2.2.4 (COLLOIDAL) COOMASSIE BLUE STAINING
.................................................................
41
2.2.2.5
IMMUNOPRECIPITATION..........................................................................................
41
2.2.2.6 BIOTIN-STREPTAVIDIN AFFINITY PURIFICATION (BIOLD PULL DOWN)
.................................
42
2.2.2.7 MASS SPECTROMETRY
ANALYSIS.............................................................................
43
2.2.2.8 RECOMBINANT PROTEIN EXPRESSION AND
PURIFICATION...........................................44
2.2.2.9 PRODUCTION OF POLYCLONAL PEPTIDE ANTIBODY
.......................................................
44
2.2.2.10 PURIFICATION OF POLYCLONAL PEPTIDE
ANTIBODY.................................................... 45
2.2.2.11 IN VITRO DECATENATION
ASSAY.............................................................................
45
2.2.2.12 IN VIVO UBIQUITYLATION
ASSAY..............................................................................46
2.2.2.13 IN VITRO TRANSCRIPTION AND IN VITRO BINDING
ASSAYS............................................46
2.2.3 METHODS IN MOLECULAR
BIOLOGY....................................................................
.
...........
47
2.2.3.1 POLYMERASE CHAIN REACTION (PCR)
..................................
.................................47
2.2.3.2 AGAROSE GEL
ELECTROPHORESIS..............................................................................48
2.2.3.3 ISOLATION AND PURIFICATION OF DNA FRAGMENTS FROM AGAROSE
GELS.....................48
2.2.3.4 RESTRICTION DIGEST OF
DNA.................................................................................
48
2.2.3.5 DEPHOSPHORYLATION OF DIGESTED VECTOR D
NA.................................................... 49
2.2.3.6 LIGATION OF DNA
FRAGMENTS................................................................................49
2.2.3.7 PREPARATION OF CHEMICALLY COMPETENT E.COLI XL1 BLUE
....................................
50
2.2.3.8 TRANSFORMATION OF COMPETENT E.COLI XL1 BLUE/ BL21-ROSETTA/
E.COLI XL10
GOLD.................................................................................................................................50
2.2.3.9 ISOLATION OF PLASMID DNA FROM E. COLI
..............................................................
50
2.2.3.10 ETHANOL PRECIPITATION OF
DNA...........................................................................51
2.2.3.11 DETERMINATION OF DNA CONCENTRATION
..............................................................
51
2.2.3.12 SYNTHESIZED DNA
FRAGMENTS...........................................................................51
3
RESULTS...................................................................................................................................53
3.1 FUNCTIONAL CHARACTERIZATION OF HUMAN FBXO28
..............................................................
53
3.1.1 FBXO28 IS AN EVOLUTIONARY CONSERVED F-BOX
PROTEIN.............................................53
3.1.2 GENERATION AND CHARACTERIZATION OF A PEPTIDE ANTIBODY AGAINST
FBXO28
.............
54
3.1.3 CELL CYCLE DISTRIBUTION OF
FBXO28............................................................................55
3.1.4 FBXO28 IS PART OF AN
SCF-COMPLEX........................................................................58
3.1.5 FBXO28 FUNCTIONS IN
MITOSIS....................................................................................59
3.1.5.1 FBXO28 IS REQUIRED FOR CENTROSOMAL
CLUSTERING................................................59
3.1.5.2 FBXO28 DEPLETION IN U20S CELLS LEADS TO MULTINUCLEATION
..............................
61
3.1.5.3 FBXO28 DEPLETION LEADS TO A PROLONGED MITOSIS INDEPENDENT OF SAC
ACTIVATION
62
3.2 IDENTIFICATION OF NOVEL FBXO28 INTERACTION
PARTNERS........................................................ 65
3.2.1 FBXO28 PULL DOWN ASSAYS TO IDENTIFY FBXO28 INTERACTION PARTNERS
......................
65
3.2.1.1 DOUBLE IMMUNOPURIFICATION OF
FLAG-HA-FBXO28..............................................65
3.2.1.2 BIOLD PULL
DOWN...............................................................................................
67
3.2.2 FBXO28 INTERACTS WITH TOPOISOMERASE II
ALPHA.....................................................70
3.2.3 FBXO28 COLOCALIZES WITH ENDOGENOUS TOPO LLA PROTEIN
........................................
72
3.2.4 ANALYSIS OF THE FUNCTIONAL INTERPLAY BETWEEN FBXO28 AND TOPO LLA
.....................73
3.2.4.1 FBXO28-TOPO LLA INTERACTION IS
PHOSPHODEPENDENT........................................74
3.2 4.2 FBXO28 ABLATION DOES NOT AFFECT TOPO LLA PHOSPHORYLATION
.......................
75
3.2.4.3 FBXO28 DOWN REGULATION OR OVEREXPRESSION HAS NO EFFECT ON TOPO
LLA PROTEIN
LEVELS..............................................................................................................................
76
3.2.4.4 FBXO28 DOES NOT UBIQUITYLATE TOPO LLA IN
VIVO.................................................77
3.2.4.5 FBXO28 INFLUENCES TOPO LLA DECATENATION ACTIVITY
..........................................
79
4
DISCUSSION.............................................................................................................................
82
4.1 FBXO28 IS A CONSERVED NUCLEAR SCF-COMPLEX
PROTEIN..................................................82
4.2 FBXO28 IS INVOLVED IN CENTROSOME CLUSTERING
................................................................
83
4.3 ROLE OF FBXO28 IN
MITOSIS...............................................................................................
85
4.3.1 FBXO28 ABLATION CAUSES
MULTINUCLEATION................................................................
85
4.3.2 ROLE OF FBXO28 IN MITOTIC
PROGRESSION....................................................................
87
4.4 IDENTIFICATION OF NOVEL FBXO28 INTERACTION
PARTNERS.........................................................89
4.4.1 FBXO28 INTERACTS WITH TOPO LLA IN A PHOSPHORYLATION-DEPENDENT
MANNER
...........
89
4.4.2 TOPO LLA MIGHT NOT BE A SUBSTRATE OF THE
SCFFBXO28-COMPLEX...............................90
4.4.3 FBXO28 REGULATES TOPO LLA DECATENATION
ACTIVITY...................................................91
4.5
PERSPECTIVES...................................................................................................................
92
4.5.1 ANALYSIS OF FBXO28 PHENOTYPES IN RESPONSE TO SIRNA-MEDIATED DOWN
REGULATION
...........................................................................................................................................
92
4.5.2 TOPO LLA AS A NOVEL FBXO28 INTERACTION PARTNER
.....................................................
94
5
REFERENCES..............................................................................................................................96
6
ANNEX....................................................................................................................................
111
6.1
ABBREVIATIONS..................................................................................................................I
L L
6.2 LIST OF
FIGURES.................................................................................................................
114
6.3 LIST OF
TABLES....................................................................................................................115
7 ACKNOWLEDGEMENTS 117
|
| any_adam_object | 1 |
| author | Schmitt, Miriam |
| author_GND | (DE-588)1130426238 |
| author_facet | Schmitt, Miriam |
| author_role | aut |
| author_sort | Schmitt, Miriam |
| author_variant | m s ms |
| building | Verbundindex |
| bvnumber | BV045313965 |
| ctrlnum | (OCoLC)985159859 (DE-599)DNB1132593174 |
| discipline | Biologie Medizin |
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| physical | III, 116 Seiten Illustrationen, Diagramme 30 cm |
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| spelling | Schmitt, Miriam Verfasser (DE-588)1130426238 aut Characterization of the SCFFbxo28 ubiquitin ligase in mitosis and in topoisomerase II alpha-dependent DNA decatenation presented by Miriam Schmitt Heidelberg 2015 III, 116 Seiten Illustrationen, Diagramme 30 cm txt rdacontent n rdamedia nc rdacarrier Im Titel ist "Fbxo28" hochgestellt. Dissertation Ruperto-Carola University of Heidelberg 2016 (DE-588)4113937-9 Hochschulschrift gnd-content B:DE-101 application/pdf http://d-nb.info/1132593174/04 Inhaltsverzeichnis DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=030700930&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Schmitt, Miriam Characterization of the SCFFbxo28 ubiquitin ligase in mitosis and in topoisomerase II alpha-dependent DNA decatenation |
| subject_GND | (DE-588)4113937-9 |
| title | Characterization of the SCFFbxo28 ubiquitin ligase in mitosis and in topoisomerase II alpha-dependent DNA decatenation |
| title_auth | Characterization of the SCFFbxo28 ubiquitin ligase in mitosis and in topoisomerase II alpha-dependent DNA decatenation |
| title_exact_search | Characterization of the SCFFbxo28 ubiquitin ligase in mitosis and in topoisomerase II alpha-dependent DNA decatenation |
| title_full | Characterization of the SCFFbxo28 ubiquitin ligase in mitosis and in topoisomerase II alpha-dependent DNA decatenation presented by Miriam Schmitt |
| title_fullStr | Characterization of the SCFFbxo28 ubiquitin ligase in mitosis and in topoisomerase II alpha-dependent DNA decatenation presented by Miriam Schmitt |
| title_full_unstemmed | Characterization of the SCFFbxo28 ubiquitin ligase in mitosis and in topoisomerase II alpha-dependent DNA decatenation presented by Miriam Schmitt |
| title_short | Characterization of the SCFFbxo28 ubiquitin ligase in mitosis and in topoisomerase II alpha-dependent DNA decatenation |
| title_sort | characterization of the scffbxo28 ubiquitin ligase in mitosis and in topoisomerase ii alpha dependent dna decatenation |
| topic_facet | Hochschulschrift |
| url | http://d-nb.info/1132593174/04 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=030700930&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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