Dynamics of human DNA topoisomerases I and II:
Gespeichert in:
| 1. Verfasser: | |
|---|---|
| Format: | Buch |
| Sprache: | English |
| Veröffentlicht: |
2002
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| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | Zsfassung in dt. Sprache. - Würzburg, Univ., Diss., 2003 |
| Beschreibung: | 123 Bl. Ill., graph. Darst. |
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| 100 | 1 | |a Christensen, Morten Overby |e Verfasser |4 aut | |
| 245 | 1 | 0 | |a Dynamics of human DNA topoisomerases I and II |c von Morten Overby Christensen |
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| 300 | |a 123 Bl. |b Ill., graph. Darst. | ||
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Datensatz im Suchindex
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|---|---|
| adam_text |
CONTENT
1. INTRODUCTION - DNA TOPOISOMERASES
1
1.1 DNA TOPOLOGY AND DNA TOPOISOMERASES
1
1.2 TYPE IB DNA TOPOISOMERASES
1.2.1 CHARACTERISTICS OF HUMAN DNA TOPOISOMERASE I
1 -2.2 DOMAIN ORGANIZATION AND PROTEIN STRUCTURE OF TOPOISOMERASE I
1 -2.3 CATALYTIC CYCLE OF HUMAN TOPOISOMERASE I
1.2.4 CAMPTOTHECIN- A DRUG TARGETING HUMAN TOPOISOMERASE I
4
4
5
7
9
1.3 TYPE 11A DNA TOPOISOMERASES
1.3.1 CHARACTERISTICS OF HUMAN TOPOISOMERASES HA AND 11(3
1.3.2 DOMAIN ORGANIZATION AND PROTEIN STRUCTURE OF TOPOISOMERASE II
1.3.3 CATALYTIC CYCLE OF EUKARYOTIC TOPOISOMERASE II
1.3.4 DRUGS TARGETING TOPOISOMERASE II
1.3.5 FUNCTION OF THE C-TERMINAL DOMAIN IN EUKARYOTIC TOPOISOMERASE II
11
11
14
17
19
20
1.4 BIOLOGICAL FUNCTIONS OF EUKARYOTIC TOPOISOMERASES I AND II
1.4.1 TRANSCRIPTION AND REPLICATION
1.4.2 CHROMOSOME STRUCTURE AND CONDENSATION
1.4.3 CHROMOSOME SEGREGATION
23
24
25
27
1.5 SUBNUCLEAR DISTRIBUTION OF MAMMALIAN TOPOISOMERASES
1.5.1 TOPOISOMERASE I
1.5.2 TOPOISOMERASE II
28
28
29
2. AIMS OF STUDY
33
3. MATERIALS AND METHODS
35
3.1 PLASMID CONSTRUCTION
3.1.1 PCR
3.1.2 GEL ELECTROPHORESIS AND RECOVERY OF DNA FROM AGAROSE GELS
3.1.3 LIGATION
3.1.4 SEQUENCING OF PLASMIDS
35
}I
38
38
38
3.2 B.COLI
STRAINS, TRANSFORMATION, AND ISOLATION OF PLASMID DNA
3.2.1 E.COLI STRAIN
3.2.2 GENERATION OF ELECTROCOMPETENT E.COLI CELLS
3.2.3 TRANSFORMATION OF E.COLI BY ELECTROPORATION
3.2.4 PLASMID PREPARATION AT A SMALL SCALE (VULGO: MINIPREPS)
3.2.5 PLASMID PREPARATION AT A LARGE SCALE (VULGO: MAXIPREPS)
38
38
39
39
39
39
3.3 MAMMALIAN CELLS
3.3.1 MAMMALIAN CELLS
3.3.2 MAINTENANCE OF MAMMALIAN CELLS
3.3.3 TRANSFECTION AND SELECTION OF 293 CELLS
40
40
40
40
3.4 WESTERN BLOTTING
3.4.1 WESTERN BLOT
41
41
3.5 BANDDEPLETION ASSAY 42
3.6 COMPLEMENTATION ASSAY 42
3.7 EXTRACTION AND IMMUNOPRECIPITATION OF ACTIVE TOPOISOMERASE II FROM
MAMMALIAN CELL NUCLEI
43
3.8 ASSESSMENT OF TOPO II ACTIVITY
44
BIBLIOGRAFISCHE INFORMATIONEN
HTTP://D-NB.INFO/968408567
CONTENT
3.9 MICROSCOPY 44
3.9.1 IMMUNOCYTOCHEMISTRY 44
3.9.2 SPREADING OF NATIVE CHROMOSOMES 45
3.9.3 FLUORESCENCE MICROSCOPY 45
3.9.4 CONFOCAL LASER SCANNING MICROSCOPY 45
39.5 PHOTOBLEACHING 46
3.9.6 KINETIC MODELLING-TOPO I 46
4. RESULTS 47
4.1 CONSTRUCTION OF BICISTRONIC EXPRESSION CONSTRUCTS 47
4.2 DYNAMICS OF HUMAN DNA TOPOISOMERASES ILA AND ILF) IN LIVING CELLS 49
4.2.1 EXPRESSION OF FULL-LENGTH TOPOISOMERASE ILA AND II(J-GFP CHIMERAS
49
4.2.2 TOPOISOMERASE ILA- AND IIP-GFP CHIMERA HAVE THE SAME ENZYMATIC
ACTIVITY AS THEIR
ENDOGENOUS COUNTERPARTS. 51
4.2.3 COLOCALIZATION OFGFP-CHIMERA WITH ENDOGENOUS TOPOISOMERASE II 53
4.2.4 DISTRIBUTION OF TOPOISOMERASE ILA AND LIP IN THE INTERPHASE
NUCLEUS 54
4.2.5 ISOFORM SPECIFIC LOCALIZATION OF TOPOISOMERASE II IN MITOSIS 55
4.2.6 ACCUMULATION OF TOPOISOMERASE ILA AT THE CENTROMERES IS LOST UPON
SISTER CHROMATID
SEPARATION 57
4.2.7 TOPOISOMERASE LIP INTERACTS WITH METAPHASE CHROMOSOMES. TOO. 58
4.2.8 MOBILITY OF TOPO ILA AND LIP IN INTERPHASE CELLS AND THE EFFECT OF
TOPO II POISONS. 59
4.2.9 TOPOISOMERASE II IS FREELY MOVING BETWEEN INTERPHASE COMPARTMENTS.
61
4.2.10 IMMOBILE FRACTIONS OF TOPOISOMERASE II DO NOT EXIST IN INTERPHASE
CELLS 63
4.2.11 MOBILITY OF TOPO ILA AT MITOSIS 64
4.2.12 AXIAL LOCALIZATION OF TOPOISOMERASE II IN CHROMOSOMES? 65
4.3 CELLULAR LOCALIZATION AND MOBILITY OF HUMAN DNA TOPOISOMERASE I 69
4.3.1 EXPRESSION OF ACTIVE GFP-TOPOISOMERASE I IN 293 CELL 69
4.3.2 COLOCALIZATION OF BIOFLUORESCENT AND ENDOGENOUS TOPOISOMERASE I 70
4.3.3 DISTRIBUTION OF TOPOISOMERASE I IN THE INTERPHASE NUCLEUS 71
4.3.4 TOPOISOMERASE I ACCUMULATES AT THE FIBRILLAR CENTERS OF THE
NUCLEOLUS 72
4.3.5 TOPOISOMERASE I IS CHROMOSOME-ASSOCIATED THROUGHOUT MITOSIS 73
4.3.6 MOBILITY OF TOPO I IN INTERPHASE 74
4.3.7 CORRELATION BETWEEN MOBILITY AND LOCALIZATION OF TOPOISOMERASE I
75
4.3.8 THE IMPACT OF CAMPTOTHECIN ON TOPOISOMERASE I MOBILITY 76
4.4 ITS NON-CONSERVED N-TERMINAL DOMAIN ANCHORS TOPOISOMERASE I AT
FIBRILLAR CENTERS OF
NUCLEOLI AND NUCLEOLAR ORGANIZER REGIONS OF MITOTIC CHROMOSOMES 79
4.4.1 EXPRESSION OF CHIMERAS OF GFP WITH N- AND C-TERMINAL DOMAINS OF
TOPOISOMERASE I 79
4.4.2 THE N-TERMINAL DOMAIN DIRECTS TOPO I TO FIBRILLAR CENTERS OF
NUCLEOLI 80
4.4.3 ON MITOTIC CHROMOSOMES, THE N-TERMINAL DOMAIN ALLOCATES
TOPOISOMERASE I TO NUCLEOLAR
ORGANIZER REGIONS 81
5. DISCUSSION 85
5.1 TOPOISOMERASE 1 AND II ARE ENTIRELY MOBILE NUCLEAR PROTEINS. 85
5.1.1 MOBILITY OF TOPOISOMERASE I AND ITS IMPLICATIONS 85
5.1.2 MOBILITY OF TOPOISOMERASE II AND ITS IMPLICATIONS 86
5.2 TOPOISOMERASE I AND II AT THE NUCLEOLUS 87
5.2.1 PUTATIVE NUCLEOLAR ROLE OF TOPOISOMERASES ILA AND P 87
5.2.2 TIGHT ASSOCIATION OF TOPOISOMERASE I WITH FCS AND NORS THROUGHOUT
THE CELL-CYCLE. 89
5.3 SPECIFIC ROLES OF TOPOISOMERASES ILA AND P DURING CELL DIVISION 90
5.4 IMMOBILISATION OF TOPOISOMERASES BY TOPOISOMERASE POISONS. 91
5.4.1 THE IMPACT OF CPT ON THE MOBILITY OF TOPOISOMERASE I 91
CONTENT
5-4-2 MOBILITY OF TOPOISOMERASES AS A MEANS TO CHARACTERIZE
TOPOISOMERASE-DIRECTED DRUGS 92
6
. SUMMARY
94
7. ZUSAMMENFASSUNG
95
8
. LIST OF ABBREVIATIONS
97
9. ACKNOWLEDGEMENTS
99
10. LIST OF PUBLICATIONS
100
11. CURRICULUM VITAE 101
12. REFERENCES 102 |
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| physical | 123 Bl. Ill., graph. Darst. |
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| spelling | Christensen, Morten Overby Verfasser aut Dynamics of human DNA topoisomerases I and II von Morten Overby Christensen 2002 123 Bl. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Zsfassung in dt. Sprache. - Würzburg, Univ., Diss., 2003 Molekulargenetik (DE-588)4039987-4 gnd rswk-swf DNS-Topoisomerasen (DE-588)4333298-5 gnd rswk-swf Mensch (DE-588)4038639-9 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Mensch (DE-588)4038639-9 s DNS-Topoisomerasen (DE-588)4333298-5 s Molekulargenetik (DE-588)4039987-4 s DE-604 DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=010243140&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Christensen, Morten Overby Dynamics of human DNA topoisomerases I and II Molekulargenetik (DE-588)4039987-4 gnd DNS-Topoisomerasen (DE-588)4333298-5 gnd Mensch (DE-588)4038639-9 gnd |
| subject_GND | (DE-588)4039987-4 (DE-588)4333298-5 (DE-588)4038639-9 (DE-588)4113937-9 |
| title | Dynamics of human DNA topoisomerases I and II |
| title_auth | Dynamics of human DNA topoisomerases I and II |
| title_exact_search | Dynamics of human DNA topoisomerases I and II |
| title_full | Dynamics of human DNA topoisomerases I and II von Morten Overby Christensen |
| title_fullStr | Dynamics of human DNA topoisomerases I and II von Morten Overby Christensen |
| title_full_unstemmed | Dynamics of human DNA topoisomerases I and II von Morten Overby Christensen |
| title_short | Dynamics of human DNA topoisomerases I and II |
| title_sort | dynamics of human dna topoisomerases i and ii |
| topic | Molekulargenetik (DE-588)4039987-4 gnd DNS-Topoisomerasen (DE-588)4333298-5 gnd Mensch (DE-588)4038639-9 gnd |
| topic_facet | Molekulargenetik DNS-Topoisomerasen Mensch Hochschulschrift |
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