Mutations at the N-terminus of scMlh2/hPms1 result in mutant MutLβ complexes that interfere with DNA mismatch repair:
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| Format: | Abschlussarbeit Buch |
| Sprache: | English |
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Heidelberg
2018
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| Online-Zugang: | Inhaltsverzeichnis Inhaltsverzeichnis |
| Beschreibung: | xviii, 130 Seiten Illustrationen, Diagramme 30 cm |
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| 100 | 1 | |a Zhao, Bo Yu |e Verfasser |0 (DE-588)1171829582 |4 aut | |
| 245 | 1 | 0 | |a Mutations at the N-terminus of scMlh2/hPms1 result in mutant MutLβ complexes that interfere with DNA mismatch repair |c presented by M.Sc. Bo Yu Zhao ; referees: Prof. Dr. Elmar Schiebel, Dr. Hans Hombauer |
| 264 | 1 | |a Heidelberg |c 2018 | |
| 300 | |a xviii, 130 Seiten |b Illustrationen, Diagramme |c 30 cm | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 502 | |b Dissertation |c Ruperto Carola University Heidelberg |d 2018 | ||
| 546 | |a Mit einer Zusammenfassung in englischer und deutscher Sprache | ||
| 655 | 7 | |0 (DE-588)4113937-9 |a Hochschulschrift |2 gnd-content | |
| 700 | 1 | |a Schiebel, Elmar |d 1960- |0 (DE-588)1034622595 |4 dgs | |
| 710 | 2 | |a Universität Heidelberg |0 (DE-588)2024349-2 |4 dgg | |
| 856 | 4 | 2 | |m B:DE-101 |q application/pdf |u http://d-nb.info/1172042284/04 |3 Inhaltsverzeichnis |
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| 943 | 1 | |a oai:aleph.bib-bvb.de:BVB01-030742021 | |
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| _version_ | 1822127855841050624 |
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| adam_text |
TABLE OF CONTENTS
CONTENTS
ACKNOWLEDGMENTS.
III
ABSTRACT.
V
ZUSAMMENFASSUNG.
VI
TABLE OF
CONTENTS.
VIII
LIST OF
TABLES.XII
LIST OF
FIGURES.XIV
LIST OF
ABBREVIATIONS.
XVI
1
INTRODUCTION.
1
1.1 DNA REPLICATION IN
EUKARYOTES.1
1.2 DNA MISMATCH
REPAIR.4
1.2.1 DNA MMR IN
PROKARYOTES.
4
1.2.1.1 DNA MMR IN ESCHERICHIA
COLI.4
1.2.1.2 DNA MMR IN OTHER
BACTERIA.
6
1.2.2 OVERVIEW OF DNA MMR IN EUKARYOTES
.
6
1.2.3 STRUCTURAL INSIGHTS INTO MISMATCH RECOGNITION BY MUTS HOMOLOGUES
.
10
1.2.4 MUTL
HOMOLOGUES.
13
1.2.4.1 FUNCTIONS OF MUTL HOMOLOGUES IN EUKARYOTES.
13
1.2.4.2 STRUCTURAL STUDIES OF MUTL HOMOLOGUES
.
14
1.2.4.2.1 GHKL FAMILY OF
ATPASES.
14
1.2.4.2.2 STRUCTURES OF MUTL
HOMOLOGUES.
15
1.2.4.2J THE NTD-DIMERIZATION CYCLE OF MUTL DIMERS
.
18
1.2.5 MECHANISMS AND MODELS OF MMR INITIATION 21
1.2.6 REGULATION OF MMR DURING DNA REPLICATION IN EUKARYOTES
.
25
1.2.6.1 THE ROLE OF PCNA IN M M
R.25
1.2.6.2 REGULATION OF MMR IN THE PRESENCE OF NUCLEOSOMES
.
26
1.2.7 THE NON-CANONICAL FUNCTIONS OF MMR
PROTEINS.27
1.3 THESIS
RATIONALE.
29
2 MATERIALS AND M
ETHODS.31
2.1 ASSAYS FOR STUDYING THE MLH2 MUTATIONS IN S. CEREVISIAE
.
31
2.1.1 YEAST
TRANSFORMATION.
31
2.1.2 PREPARATION OF AMINO ACID DROPOUT MEDIA AND PLATES
.
32
2.1.3 GENETIC SCREEN TO IDENTIFY MLH2 DOMINANT MUTATIONS THAT INTERFERE
WITH MMR
IN S.
CEREVISIAE.33
2.1.4 SUPPRESSION OF MUTATOR PHENOTYPES CAUSED BY THE MLH2 DOMINANT
MUTATIONS BY
OVEREXPRESSION OF POL30 AND SCP M SL
.
34
2.1.4.1 PATCH
TEST.34
2.1.4.2 FLUCTUATION
ANALYSIS.
34
2.1.5 PATCH TEST FOR YEAST STRAINS THAT EXPRESS MLH2 AND MLH3 M UTANTS
.
38
2.2 ASSAYS FOR STUDYING HUMANPM SL MUTATIONS IN MAMMALIAN TISSUE
CULTURE.42
2.2.1 MAINTENANCE OF CELL LINES AND GENERAL TRANSFECTION PROCEDURES
.
42
2.2.2 MICROSATELLITE INSTABILITY (MSI) FLUORESCENCE REPORTER ASSAY
.
43
2.2.2.1 CONSTRUCTION OF MSI FLUORESCENT REPORTER PLASMIDS
.
43
2.2.2.2 GENERATION OF HEK293 FLP-IN T-REX MSI REPORTER CELL LINES
.
45
2.2.2.3 DOWNREGULATION OF HPMSL OR HMLHL BY SHRNA USING THE
PSUPERIOR.PURO VECTOR
.
47
2.2.2.4 CONSTRUCTION OF PCDNA5/FRT/TO PLASMIDS THAT CO-EXPRESS HMLHL
AND HPMSL IN EITHER THE HPMSL-P2A-HMLHL OR HMLHL-P2A-HPMSL
ORIENTATION.
48
2.2.2.5 CONSTRUCTION OF PSUPERIOR.PURO-ZZ.PMS7SHRNA PLASMIDS THAT CO
EXPRESS HMLHL AND H P M SL
.
51
2.2.2.6 MSI ANALYSIS BY FLOW
CYTOMETRY.
56
IX
2.2.3 GENERATION OF KNOCKOUT CELL LINES USING
CRISPR-CAS9.56
2.2.3.1 CONSTRUCTION OF PLENTICRISPR-CAS9 SGRNAS AGAINST HMLHL,
HPMSL AND
HEXOI.
56
2.2.3.2 NUCLEOFECTION AND GENOMIC DNA
EXTRACTION.58
2.2.3.3 T7 ENDONUCLEASE
ASSAY.
58
2.23.4 GENERATION OF (H)MLHLA, (H)PMSIA, AND (H)EXOIA CELL LINES
.
59
2.2.4 CONSTRUCTION OF HOMOLOGOUS RECOMBINATION TEMPLATE PLASMIDS FOR
GENERATING
THE (H) MLHL-A21P AND (H)
PMSL-SM P POINT MUTATIONS IN THE HAP1 CELL LINE
.
60
2.2.5 GENERATION OF HAP 1 MLHL-A21P AND HAP1 PMSL-S14P CELL LINES
.
63
2.2.5.1 GENOMIC DNA ISOLATION IN 96-WELL
PLATES.63
2.2.5.2 GENERATION OF THE MAPI MLHL-A21P AND
PMSL-S14P CELL LINES USING
CRISPR-CAS9
.
64
2.2.53 QUALITATIVE AND QUANTITATIVE HYPOXANTHINE-GUANINE
PHOSPHORIBOSYLTRANSFERASE (HPRT)
ASSAY.67
2.3 IN VITRO DNA MISMATCH REPAIR
ASSAY.
68
2.3.1 GENERATION OF G-T AND LT-INSERT DNA
HETERODUPLEX.68
23.1.1 EXTRACTION OF SSCDNA WITH R408 HELPER PHAGE
.
68
23.1.2 GENERATION OF DNA HETERODUPLEX
.
69
2.3.2 PREPARATION OF NUCLEAR EXTRACT FROM MAMMALIAN
CELLS.70
2.3.3 IN VITRO MMR REPAIR
ASSAY.
71
3
RESULTS.72
3.1 IDENTIFICATION AND CHARACTERIZATION OF MLH2 DOMINANT MUTATIONS IN S.
CEREVISIAE.72
3.1.1 OVEREXPRESSION OF POL30 OR SCPMS 1 SUPPRESS MUTATOR PHENOTYPES
CAUSED BY
DOMINANT MLH2 MUTATIONS IN S. CEREVISIAE
.
72
3.1.2 MUTATIONS IN OTHER CONSERVED RESIDUES IN THE NTDS OF SCMLH2 AND
SCMLH3 DO
NOT RESULT IN MUTATOR
PHENOTYPES.76
3.2 CHARACTERIZATION OF (H)PMSL MUTATIONS BY A MSI FLUORESCENCE REPORTER
ASSAY IN
MAMMALIAN
CELLS.
79
3.2.1 GENERATION OF HEK293 FLP T-REX MSI FLUORESCENCE REPORTER CELL
LINES 80
3.2.2 HMLHL AND HPMSL ARE EXPRESSED AT A HIGHER LEVEL FROM THE
HMLHL-P2A-
HPMSL ORIENTATION THAN FROM THE HPMSL-P2A-HMLHL ORIENTATION
.
83
3.2.3 CELLS EXPRESSING (H)PMSL MUTANTS SHOW SIMILAR LEVEL OF MSI AS
CELLS
EXPRESSING WT HP M
SL.85
3.3 CHARACTERIZATION OF THE (H)
PMSL-SM P AND (H) MLHL-A21P MUTATIONS IN THE HAP1 CELL
LINE.
89
3.3.1 GENERATION OF (H) MLHLA AND (H)PMSLA CELL
LINES.90
3.3.2 THE (H) M\HL-A21P MUTATION AND (H)PMSL-S14P MUTATION BOTH CAUSED
MUTATOR
PHENOTYPES IN THE HAP1 CELL
LINE.93
3.4 ESTABLISHMENT OF AN IN VITRO MMR
ASSAY.96
3.5 GENERATION OF A HEK293 FLP T-REX EXOIA CELL LINE
.
98
4 DISCUSSIONS AND FUTURE
DIRECTIONS.
100
APPENDICES.
110
REFERENCES.
112 |
| any_adam_object | 1 |
| author | Zhao, Bo Yu |
| author_GND | (DE-588)1171829582 (DE-588)1034622595 |
| author_facet | Zhao, Bo Yu |
| author_role | aut |
| author_sort | Zhao, Bo Yu |
| author_variant | b y z by byz |
| building | Verbundindex |
| bvnumber | BV045355424 |
| ctrlnum | (OCoLC)1136480105 (DE-599)BSZ513864180 |
| discipline | Biologie |
| format | Thesis Book |
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| id | DE-604.BV045355424 |
| illustrated | Illustrated |
| indexdate | 2025-01-24T11:02:16Z |
| institution | BVB |
| institution_GND | (DE-588)2024349-2 |
| language | English |
| oai_aleph_id | oai:aleph.bib-bvb.de:BVB01-030742021 |
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| physical | xviii, 130 Seiten Illustrationen, Diagramme 30 cm |
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| spelling | Zhao, Bo Yu Verfasser (DE-588)1171829582 aut Mutations at the N-terminus of scMlh2/hPms1 result in mutant MutLβ complexes that interfere with DNA mismatch repair presented by M.Sc. Bo Yu Zhao ; referees: Prof. Dr. Elmar Schiebel, Dr. Hans Hombauer Heidelberg 2018 xviii, 130 Seiten Illustrationen, Diagramme 30 cm txt rdacontent n rdamedia nc rdacarrier Dissertation Ruperto Carola University Heidelberg 2018 Mit einer Zusammenfassung in englischer und deutscher Sprache (DE-588)4113937-9 Hochschulschrift gnd-content Schiebel, Elmar 1960- (DE-588)1034622595 dgs Universität Heidelberg (DE-588)2024349-2 dgg B:DE-101 application/pdf http://d-nb.info/1172042284/04 Inhaltsverzeichnis DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=030742021&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Zhao, Bo Yu Mutations at the N-terminus of scMlh2/hPms1 result in mutant MutLβ complexes that interfere with DNA mismatch repair |
| subject_GND | (DE-588)4113937-9 |
| title | Mutations at the N-terminus of scMlh2/hPms1 result in mutant MutLβ complexes that interfere with DNA mismatch repair |
| title_auth | Mutations at the N-terminus of scMlh2/hPms1 result in mutant MutLβ complexes that interfere with DNA mismatch repair |
| title_exact_search | Mutations at the N-terminus of scMlh2/hPms1 result in mutant MutLβ complexes that interfere with DNA mismatch repair |
| title_full | Mutations at the N-terminus of scMlh2/hPms1 result in mutant MutLβ complexes that interfere with DNA mismatch repair presented by M.Sc. Bo Yu Zhao ; referees: Prof. Dr. Elmar Schiebel, Dr. Hans Hombauer |
| title_fullStr | Mutations at the N-terminus of scMlh2/hPms1 result in mutant MutLβ complexes that interfere with DNA mismatch repair presented by M.Sc. Bo Yu Zhao ; referees: Prof. Dr. Elmar Schiebel, Dr. Hans Hombauer |
| title_full_unstemmed | Mutations at the N-terminus of scMlh2/hPms1 result in mutant MutLβ complexes that interfere with DNA mismatch repair presented by M.Sc. Bo Yu Zhao ; referees: Prof. Dr. Elmar Schiebel, Dr. Hans Hombauer |
| title_short | Mutations at the N-terminus of scMlh2/hPms1 result in mutant MutLβ complexes that interfere with DNA mismatch repair |
| title_sort | mutations at the n terminus of scmlh2 hpms1 result in mutant mutlβ complexes that interfere with dna mismatch repair |
| topic_facet | Hochschulschrift |
| url | http://d-nb.info/1172042284/04 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=030742021&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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