DNA Ligase I activity dictates a window of time for Mismatch Repair strand discrimination in Saccharomyces cerevisiae:
Gespeichert in:
| 1. Verfasser: | |
|---|---|
| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
Heidelberg
2020
|
| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis Inhaltsverzeichnis |
| Beschreibung: | xii, 106 Seiten Illustrationen |
Internformat
MARC
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| 100 | 1 | |a Kolodziejczak, Anna |d 1990- |e Verfasser |0 (DE-588)1209238624 |4 aut | |
| 245 | 1 | 0 | |a DNA Ligase I activity dictates a window of time for Mismatch Repair strand discrimination in Saccharomyces cerevisiae |c presented by M.Sc. Anna Kolodziejczak |
| 264 | 1 | |a Heidelberg |c 2020 | |
| 300 | |a xii, 106 Seiten |b Illustrationen | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 502 | |b Dissertation |c Ruperto Carola University Heidelberg |d 2020 | ||
| 655 | 7 | |0 (DE-588)4113937-9 |a Hochschulschrift |2 gnd-content | |
| 856 | 4 | 2 | |m B:DE-101 |q application/pdf |u https://d-nb.info/1214264905/04 |3 Inhaltsverzeichnis |
| 856 | 4 | 2 | |m DNB Datenaustausch |q application/pdf |u http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=032229918&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |3 Inhaltsverzeichnis |
| 999 | |a oai:aleph.bib-bvb.de:BVB01-032229918 | ||
Datensatz im Suchindex
| _version_ | 1804181635254452224 |
|---|---|
| adam_text | TABLE
OF
CONTENTS
SUMMARY
I
ZUSAMMENFASSUNG
II
PREFACE
IV
TABLE
OF
CONTENTS
LIST
OF
FIGURES
VIII
LIST
OF
TABLES
IX
LIST
OF
ABBREVIATIONS
X
1.
INTRODUCTION
1
1.1
DNA
REPLICATION
1
1.2
OKAZAKI
FRAGMENT
MATURATION
3
1.2.1
PROLIFERATING
CELL
NUCLEAR
ANTIGEN
(PCNA)
4
1.2.2
FLAP
ENDONUCLEASE
RAD27
6
1.2.3
DNA
LIGASE
I
(CDC9)
7
1.3
DNA
MISMATCH
REPAIR
12
1.4
MODELS
FOR
MMR
STRAND
DISCRIMINATION
16
1.5
URACIL
AS
A
SOURCE
OF
DNA
DAMAGE
18
2.
MATERIALS
22
2.1
EQUIPMENT
22
2.2
SOFTWARE
24
2.3
CONSUMABLES
24
2.4
KITS
25
2.5 CHEMICALS
AND
REAGENTS
26
2.6
MARKERS
FOR
ELECTROPHORESIS
28
2.7
OLIGONUCLEOTIDES
28
2.8
PLASMIDS
30
2.9 ENZYMES
31
2.10
ANTIBODIES
32
2.11
BUFFERS
AND
SOLUTIONS
32
2.12
MEDIA
35
2.13
E.
COLI
STRAINS
36
2.14
S.
CEREVISIAE
STRAINS
36
3.
METHODS
40
3.1
MOLECULAR
BIOLOGY
TECHNIQUES40
3.1.1
AGAROSE
GEL
ELECTROPHORESIS
40
3.1.2
POLYMERASE
CHAIN
REACTION
(PCR)
40
V
3.1.3
COLONY
POLYMERASE
CHAIN
REACTION
..................................................................................
41
3.1.4
GENERATION
OF
ELECTROCOMPETENT
CELLS
(TOP
10
F
*
)
........................................................
41
3.1.5
PLASMID
TRANSFORMATION
IN
E.
COLI
....................................................................................
41
3.1.6
WESTERN
BLOT
....................................................................................................................
42
3.2
S.
CEREVISIAE
METHODS
........................................................................................................
43
3.2.1
GROWTH
CONDITIONS
...........................................................................................................
43
3.2.2
STRAIN
CONSTRUCTION
.............................................................................
43
3.2.3
GENERATION
OF
YEAST
COMPETENT
CELLS
..............................................................................
43
3.2.4
YEAST
TRANSFORMATION
.......................................................................................................
43
3.2.5
PURIFICATION
OF
GENOMIC
DNA
...........................................................................................
44
3.2.6
PLASMID
RESCUE
FROM
YEAST
CELLS
.....................................................................................
44
3.2.7
YEAST
CRUDE
CELL
LYSATES
..................................................................................................
45
3.2.8
DETERMINATION
OF
MUTATION
RATES
IN
YEAST
CELLS
...............................................................
45
3.2.9
CAN1
MUTATION
SPECTRA
ANALYSIS
.....................................................................................
45
3.2.10
OVEREXPRESSION
SCREEN
IN
AN
EXO1
-DEFICIENT
BACKGROUND
.........................................
45
3.2.11
DETERMINATION
OF
DOUBLING
TIMES
OF
YEAST
CULTURES
....................................................
46
3.2.12
YEAST-TWO-HYBRID
ASSAY
............................................................................................
46
3.3
MAMMALIAN
CELL
CULTURE
METHODS
......................................................................................
47
3.3.1
MAMMALIAN
CELLS
GROWTH
CONDITIONS
..............................................................................
47
3.3.2
CLONING
AND
VERIFICATION
OF
SGRNA
TARGETING
HUMAN
UNG
GENE
INTO
THE
CRISPR-CAS9
PLASMID47
3.3.3
TRANSFECTION
OF
MAMMALIAN
CELLS
....................................................................................
47
3.3.4
SELECTION
AND
VERIFICATION
OF
HCT1
16
UNGA
CLONES
........................................................
48
3.3.5
GENOMIC
DNA
ISOLATION
FROM
HCT116
CELL
LINES
..............................................................
50
3.3.6
CYTOTOXICITY
ASSAY
...........................................................................................................
50
3.3.7
URACIL
ACCUMULATION
ASSAY
..............................................................................................
50
4.
RESULTS
.........................................................................................................................................
51
4.1
CDC9-OVEREXPRESSION
IN
EXO1-DEFICIENT
STRAINS
CAUSES
INCREASED
MUTATION
RATES
...........
51
4.2
INCREASED
OCCURRENCE
OF
FRAMESHIFT
MUTATIONS
AS
A
RESULT
OF
DNA
LIGASE
OVEREXPRESSION
IN
EXO1-MUTATED
BACKGROUND
..............................................................................................................
54
4.3
CDC9
OVEREXPRESSION
INTERFERES
WITH
EXO1-DEPENDENT
AND
EXO1-INDEPENDENT
MMR
.......
57
4.4 CDC9-OVEREXPRESSION INTERFERES
WITH
MMR
AT
BOTH
LEADING
AND
LAGGING
STRAND
.................
58
4.5 CDC9-OVEREXPRESSION
DOES
NOT
ACTIVATE
THE
DNA
DAMAGE
CHECKPOINT
...............................
65
4.6 OVEREXPRESSION
SCREEN
IN
AN
EXO1A
BACKGROUND:
MLH2
OVEREXPRESSION
CAUSES
A
MUTATOR
PHENOTYPE
........................................................................................................................................
65
4.7
EXPRESSION
OF
DNA
LIGASE
I
IN
G2/M
PHASE
OR
A
CDC9-FFAA
MUTANT
ALLELE
RESCUES
THE
MUTATOR
PHENOTYPE
OF
THE
G2/M-PMS1
STRAIN
.................................................................................
67
VI
4.8
YEAST
TWO
HYBRID
ANALYSIS
REVEALS
THAT
THE
INTERACTION
BETWEEN
MLH1
AND
PMS1
IS
NOT
AFFECTED
BY
THE
PMS1-E707K
MUTATION
............................................................................................
69
4.9
THE
DUT1-1
MUTATION
RESULTS
IN
A
GROWTH
DEFECT
BUT
DOES
NOT
COMPROMISE
GENOME
STABILITY.
70
4.10 UNG-DEFICIENT
HCT116
CANCER
CELLS
SHOW
INCREASED
URACIL
INCORPORATION
AND
SENSITIVITY
TO
METHOTREXATE
....................................................................................................................................
71
5.
DISCUSSION
..................................................................................................................................
74
5.1
DNA
LIGASE
I
OVEREXPRESSION
CAUSES
A
SYNERGISTIC
INCREASE
IN
THE
MUTATION
RATES
IN
CELLS
WITH
PARTIALLY
COMPROMISED
MMR....................................................................................................
74
5.2
INCREASED
EXPRESSION
OF
CDC9
AFFECTS
THE
LEADING
AND
THE
LAGGING
STRAND
TO
THE
SAME
EXTENT.
77
5.3
DNA
NICKS
ACT
AS
DNA
STRAND
DISCRIMINATION
SIGNAL
FOR
MISMATCH
REPAIR.........................
79
5.4
DECREASED
DUT1
ACTIVITY
IN
THE
DUT1-1
MUTANT
DOES
NOT
RESULT
IN
GENOMIC
INSTABILITY
IN
S.
CEREVISIAE
.........................................................................................................................................
82
5.5
CONCLUDING
REMARKS
AND
FUTURE
OUTLOOK
.............................................................................
82
6.
BIBLIOGRAPHY
..............................................................................................................................
84
7.
SUPPLEMENTARY
DATA
..................................................................................................................
98
7.1
ACKNOWLEDGEMENTS
..........................................................................................................
106
VII
|
| adam_txt |
TABLE
OF
CONTENTS
SUMMARY
I
ZUSAMMENFASSUNG
II
PREFACE
IV
TABLE
OF
CONTENTS
LIST
OF
FIGURES
VIII
LIST
OF
TABLES
IX
LIST
OF
ABBREVIATIONS
X
1.
INTRODUCTION
1
1.1
DNA
REPLICATION
1
1.2
OKAZAKI
FRAGMENT
MATURATION
3
1.2.1
PROLIFERATING
CELL
NUCLEAR
ANTIGEN
(PCNA)
4
1.2.2
FLAP
ENDONUCLEASE
RAD27
6
1.2.3
DNA
LIGASE
I
(CDC9)
7
1.3
DNA
MISMATCH
REPAIR
12
1.4
MODELS
FOR
MMR
STRAND
DISCRIMINATION
16
1.5
URACIL
AS
A
SOURCE
OF
DNA
DAMAGE
18
2.
MATERIALS
22
2.1
EQUIPMENT
22
2.2
SOFTWARE
24
2.3
CONSUMABLES
24
2.4
KITS
25
2.5 CHEMICALS
AND
REAGENTS
26
2.6
MARKERS
FOR
ELECTROPHORESIS
28
2.7
OLIGONUCLEOTIDES
28
2.8
PLASMIDS
30
2.9 ENZYMES
31
2.10
ANTIBODIES
32
2.11
BUFFERS
AND
SOLUTIONS
32
2.12
MEDIA
35
2.13
E.
COLI
STRAINS
36
2.14
S.
CEREVISIAE
STRAINS
36
3.
METHODS
40
3.1
MOLECULAR
BIOLOGY
TECHNIQUES40
3.1.1
AGAROSE
GEL
ELECTROPHORESIS
40
3.1.2
POLYMERASE
CHAIN
REACTION
(PCR)
40
V
3.1.3
COLONY
POLYMERASE
CHAIN
REACTION
.
41
3.1.4
GENERATION
OF
ELECTROCOMPETENT
CELLS
(TOP
10
F
*
)
.
41
3.1.5
PLASMID
TRANSFORMATION
IN
E.
COLI
.
41
3.1.6
WESTERN
BLOT
.
42
3.2
S.
CEREVISIAE
METHODS
.
43
3.2.1
GROWTH
CONDITIONS
.
43
3.2.2
STRAIN
CONSTRUCTION
.
43
3.2.3
GENERATION
OF
YEAST
COMPETENT
CELLS
.
43
3.2.4
YEAST
TRANSFORMATION
.
43
3.2.5
PURIFICATION
OF
GENOMIC
DNA
.
44
3.2.6
PLASMID
RESCUE
FROM
YEAST
CELLS
.
44
3.2.7
YEAST
CRUDE
CELL
LYSATES
.
45
3.2.8
DETERMINATION
OF
MUTATION
RATES
IN
YEAST
CELLS
.
45
3.2.9
CAN1
MUTATION
SPECTRA
ANALYSIS
.
45
3.2.10
OVEREXPRESSION
SCREEN
IN
AN
EXO1
-DEFICIENT
BACKGROUND
.
45
3.2.11
DETERMINATION
OF
DOUBLING
TIMES
OF
YEAST
CULTURES
.
46
3.2.12
YEAST-TWO-HYBRID
ASSAY
.
46
3.3
MAMMALIAN
CELL
CULTURE
METHODS
.
47
3.3.1
MAMMALIAN
CELLS
GROWTH
CONDITIONS
.
47
3.3.2
CLONING
AND
VERIFICATION
OF
SGRNA
TARGETING
HUMAN
UNG
GENE
INTO
THE
CRISPR-CAS9
PLASMID47
3.3.3
TRANSFECTION
OF
MAMMALIAN
CELLS
.
47
3.3.4
SELECTION
AND
VERIFICATION
OF
HCT1
16
UNGA
CLONES
.
48
3.3.5
GENOMIC
DNA
ISOLATION
FROM
HCT116
CELL
LINES
.
50
3.3.6
CYTOTOXICITY
ASSAY
.
50
3.3.7
URACIL
ACCUMULATION
ASSAY
.
50
4.
RESULTS
.
51
4.1
CDC9-OVEREXPRESSION
IN
EXO1-DEFICIENT
STRAINS
CAUSES
INCREASED
MUTATION
RATES
.
51
4.2
INCREASED
OCCURRENCE
OF
FRAMESHIFT
MUTATIONS
AS
A
RESULT
OF
DNA
LIGASE
OVEREXPRESSION
IN
EXO1-MUTATED
BACKGROUND
.
54
4.3
CDC9
OVEREXPRESSION
INTERFERES
WITH
EXO1-DEPENDENT
AND
EXO1-INDEPENDENT
MMR
.
57
4.4 CDC9-OVEREXPRESSION INTERFERES
WITH
MMR
AT
BOTH
LEADING
AND
LAGGING
STRAND
.
58
4.5 CDC9-OVEREXPRESSION
DOES
NOT
ACTIVATE
THE
DNA
DAMAGE
CHECKPOINT
.
65
4.6 OVEREXPRESSION
SCREEN
IN
AN
EXO1A
BACKGROUND:
MLH2
OVEREXPRESSION
CAUSES
A
MUTATOR
PHENOTYPE
.
65
4.7
EXPRESSION
OF
DNA
LIGASE
I
IN
G2/M
PHASE
OR
A
CDC9-FFAA
MUTANT
ALLELE
RESCUES
THE
MUTATOR
PHENOTYPE
OF
THE
G2/M-PMS1
STRAIN
.
67
VI
4.8
YEAST
TWO
HYBRID
ANALYSIS
REVEALS
THAT
THE
INTERACTION
BETWEEN
MLH1
AND
PMS1
IS
NOT
AFFECTED
BY
THE
PMS1-E707K
MUTATION
.
69
4.9
THE
DUT1-1
MUTATION
RESULTS
IN
A
GROWTH
DEFECT
BUT
DOES
NOT
COMPROMISE
GENOME
STABILITY.
70
4.10 UNG-DEFICIENT
HCT116
CANCER
CELLS
SHOW
INCREASED
URACIL
INCORPORATION
AND
SENSITIVITY
TO
METHOTREXATE
.
71
5.
DISCUSSION
.
74
5.1
DNA
LIGASE
I
OVEREXPRESSION
CAUSES
A
SYNERGISTIC
INCREASE
IN
THE
MUTATION
RATES
IN
CELLS
WITH
PARTIALLY
COMPROMISED
MMR.
74
5.2
INCREASED
EXPRESSION
OF
CDC9
AFFECTS
THE
LEADING
AND
THE
LAGGING
STRAND
TO
THE
SAME
EXTENT.
77
5.3
DNA
NICKS
ACT
AS
DNA
STRAND
DISCRIMINATION
SIGNAL
FOR
MISMATCH
REPAIR.
79
5.4
DECREASED
DUT1
ACTIVITY
IN
THE
DUT1-1
MUTANT
DOES
NOT
RESULT
IN
GENOMIC
INSTABILITY
IN
S.
CEREVISIAE
.
82
5.5
CONCLUDING
REMARKS
AND
FUTURE
OUTLOOK
.
82
6.
BIBLIOGRAPHY
.
84
7.
SUPPLEMENTARY
DATA
.
98
7.1
ACKNOWLEDGEMENTS
.
106
VII |
| any_adam_object | 1 |
| any_adam_object_boolean | 1 |
| author | Kolodziejczak, Anna 1990- |
| author_GND | (DE-588)1209238624 |
| author_facet | Kolodziejczak, Anna 1990- |
| author_role | aut |
| author_sort | Kolodziejczak, Anna 1990- |
| author_variant | a k ak |
| building | Verbundindex |
| bvnumber | BV046821565 |
| ctrlnum | (OCoLC)1190913059 (DE-599)KXP1703212142 |
| discipline | Biologie |
| discipline_str_mv | Biologie |
| format | Thesis Book |
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| genre | (DE-588)4113937-9 Hochschulschrift gnd-content |
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| id | DE-604.BV046821565 |
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| index_date | 2024-07-03T15:02:14Z |
| indexdate | 2024-07-10T08:54:47Z |
| institution | BVB |
| language | English |
| oai_aleph_id | oai:aleph.bib-bvb.de:BVB01-032229918 |
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| physical | xii, 106 Seiten Illustrationen |
| publishDate | 2020 |
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| publishDateSort | 2020 |
| record_format | marc |
| spelling | Kolodziejczak, Anna 1990- Verfasser (DE-588)1209238624 aut DNA Ligase I activity dictates a window of time for Mismatch Repair strand discrimination in Saccharomyces cerevisiae presented by M.Sc. Anna Kolodziejczak Heidelberg 2020 xii, 106 Seiten Illustrationen txt rdacontent n rdamedia nc rdacarrier Dissertation Ruperto Carola University Heidelberg 2020 (DE-588)4113937-9 Hochschulschrift gnd-content B:DE-101 application/pdf https://d-nb.info/1214264905/04 Inhaltsverzeichnis DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=032229918&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Kolodziejczak, Anna 1990- DNA Ligase I activity dictates a window of time for Mismatch Repair strand discrimination in Saccharomyces cerevisiae |
| subject_GND | (DE-588)4113937-9 |
| title | DNA Ligase I activity dictates a window of time for Mismatch Repair strand discrimination in Saccharomyces cerevisiae |
| title_auth | DNA Ligase I activity dictates a window of time for Mismatch Repair strand discrimination in Saccharomyces cerevisiae |
| title_exact_search | DNA Ligase I activity dictates a window of time for Mismatch Repair strand discrimination in Saccharomyces cerevisiae |
| title_exact_search_txtP | DNA Ligase I activity dictates a window of time for Mismatch Repair strand discrimination in Saccharomyces cerevisiae |
| title_full | DNA Ligase I activity dictates a window of time for Mismatch Repair strand discrimination in Saccharomyces cerevisiae presented by M.Sc. Anna Kolodziejczak |
| title_fullStr | DNA Ligase I activity dictates a window of time for Mismatch Repair strand discrimination in Saccharomyces cerevisiae presented by M.Sc. Anna Kolodziejczak |
| title_full_unstemmed | DNA Ligase I activity dictates a window of time for Mismatch Repair strand discrimination in Saccharomyces cerevisiae presented by M.Sc. Anna Kolodziejczak |
| title_short | DNA Ligase I activity dictates a window of time for Mismatch Repair strand discrimination in Saccharomyces cerevisiae |
| title_sort | dna ligase i activity dictates a window of time for mismatch repair strand discrimination in saccharomyces cerevisiae |
| topic_facet | Hochschulschrift |
| url | https://d-nb.info/1214264905/04 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=032229918&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT kolodziejczakanna dnaligaseiactivitydictatesawindowoftimeformismatchrepairstranddiscriminationinsaccharomycescerevisiae |
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