Molecular basis for product-specificity of DOT1 methyltransferases in Trypanosoma brucei:
Gespeichert in:
| 1. Verfasser: | |
|---|---|
| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
2014
|
| Schlagworte: | |
| Online-Zugang: | Volltext Inhaltsverzeichnis |
| Beschreibung: | 77 Bl. Ill., graph. Darst. 30 cm |
Internformat
MARC
| LEADER | 00000nam a2200000 c 4500 | ||
|---|---|---|---|
| 001 | BV042217327 | ||
| 003 | DE-604 | ||
| 005 | 20150113 | ||
| 007 | t | ||
| 008 | 141201s2014 gw ad|| m||| 00||| eng d | ||
| 015 | |a 14,H11 |2 dnb | ||
| 016 | 7 | |a 1058765655 |2 DE-101 | |
| 035 | |a (OCoLC)898031986 | ||
| 035 | |a (DE-599)DNB1058765655 | ||
| 040 | |a DE-604 |b ger |e rakddb | ||
| 041 | 0 | |a eng | |
| 044 | |a gw |c XA-DE | ||
| 049 | |a DE-12 |a DE-384 |a DE-473 |a DE-703 |a DE-1051 |a DE-824 |a DE-29 |a DE-91 |a DE-19 |a DE-1049 |a DE-92 |a DE-739 |a DE-898 |a DE-355 |a DE-706 |a DE-20 |a DE-1102 | ||
| 084 | |a 570 |2 sdnb | ||
| 084 | |a 610 |2 sdnb | ||
| 100 | 1 | |a Dindar, Gülcin |d 1983- |e Verfasser |0 (DE-588)1059083337 |4 aut | |
| 245 | 1 | 0 | |a Molecular basis for product-specificity of DOT1 methyltransferases in Trypanosoma brucei |c vorgelegt von Gülcin Dindar |
| 246 | 1 | 3 | |a Die molekularen Grundlagen der Produktspezifität von DOT1 Methyltransferasen in Trypanosoma brucei |
| 264 | 1 | |c 2014 | |
| 300 | |a 77 Bl. |b Ill., graph. Darst. |c 30 cm | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 502 | |a Würzburg, Univ., Diss., 2014 | ||
| 546 | |a Zsfassung in dt. und engl. Sprache | ||
| 650 | 0 | 7 | |a Trypanosoma brucei |0 (DE-588)4186350-1 |2 gnd |9 rswk-swf |
| 650 | 0 | 7 | |a Chromatin |0 (DE-588)4010152-6 |2 gnd |9 rswk-swf |
| 650 | 0 | 7 | |a Histon-Methyltransferase |0 (DE-588)4820080-3 |2 gnd |9 rswk-swf |
| 655 | 7 | |0 (DE-588)4113937-9 |a Hochschulschrift |2 gnd-content | |
| 689 | 0 | 0 | |a Trypanosoma brucei |0 (DE-588)4186350-1 |D s |
| 689 | 0 | 1 | |a Histon-Methyltransferase |0 (DE-588)4820080-3 |D s |
| 689 | 0 | 2 | |a Chromatin |0 (DE-588)4010152-6 |D s |
| 689 | 0 | |5 DE-604 | |
| 776 | 0 | 8 | |i Erscheint auch als |n Online-Ausgabe |o urn:nbn:de:bvb:20-opus-102524 |
| 856 | 4 | 1 | |u https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-102524 |x Resolving-System |z kostenfrei |3 Volltext |
| 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=027655903&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |3 Inhaltsverzeichnis |
| 912 | |a ebook | ||
| 999 | |a oai:aleph.bib-bvb.de:BVB01-027655903 | ||
Datensatz im Suchindex
| _version_ | 1804152742744162304 |
|---|---|
| adam_text | MOLECULAR BASIS
FOR PRODUCT-SPECIFICITY
OF
DOT1
METHYLTRANSFERASES IN
TRYPANOSOMA BRUCEI
DISSERTATION ZUR ERLANGUNG DES
NATURWISSENSCHAFTLICHEN DOKTORGRADES
DER JULIUS-MAXIMILIANS-UNIVERSITAT WURZBURG
VORGELEGT VON
GULCIN DINDAR
AUS MUNCHEN
WURZBURG 2014
HTTP://D-NB.INFO/1058765655
TABLE OF CONTENTS
TABLE OF CONTENTS
ACKNOWLEDGMENTS 4
ZUSAMMENFASSUNG 5
ABSTRACT 6
ABBREVIATIONS 7
1 INTRODUCTION 10
1.1 TRYPANOSOMA BRUCEI 10
1.2 CHROMATIN STRUCTURE 11
1.2.1 CORE HISTONES AND THE NUCIEOSOME 11
1.2.2 HISTONE VARIANTS 12
1.3 CHROMATIN ASSEMBLY AND DYNAMICS 13
1.4 POSTTRANSLATIONAL HISTONE MODIFICATIONS 14
1.4.1 HISTONE TAIL MODIFICATIONS 14
1.4.2 HISTONE MODIFICATIONS IN T. BRUCEI 15
1.5 HISTONE LYSINE METHYLATION 16
1.6 SET-DOMAIN HISTONE LYSINE METHYLTRANSFERASES 17
1.7 DOT1
METHYLTRANSFERASES 17
1.7.1 OVERVIEW 17
1.7.2 THE CONSERVED DOT1 CORE 18
1.7.3 SAM-BINDING POCKET 20
1.7.4 THE SUBSTRATE LYSINE-BINDING POCKET 21
1.8 MOTIVATION 21
2 MATERIALS AND METHODS 23
2.1 MOLECULAR CLONING 23
2.1.1 EXPRESSION VECTORS AND BACTERIAL STRAINS 23
2.1.2 BACTERIAL GROWTH 23
2.1.3 POLYMERASE CHAIN REACTION (PCR) 23
2.1.4 ENZYMATIC DIGESTION OF DNA 25
2.1.5 AGAROSE GEL ELECTROPHORESIS AND GEL EXTRACTION 25
2.1.6 DNA CONCENTRATION DETERMINATION 25
2.1.7 DNA LIGATION 25
2.1.8 SITE DIRECTED MUTAGENESIS 26
2.1.9 ETHANOL PRECIPITATION 26
2.1.10 PREPARATION OF MAGNESIUM CHLORIDE COMPETENT E. COLI CELLS 26
2.1.11 DNA TRANSFORMATION INTO COMPETENT E. COLI CELLS 26
2.1.12 PLASMID ISOLATION 27
TABLE OF CONTENTS
2.1.13 DNA SEQUENCING 27
2.2 PROTEIN SAMPLE ANALYSIS 27
2.2.1 TRICHLOROACETIC ACID (TCA) PROTEIN PRECIPITATION 27
2.2.2 SODIUM DODECYL SULFATE POLYACRYLAMIDE GEL ELECTROPHORESIS
(SDS-PAGE) 27
2.2.3 PROTEIN GEL STAINING 27
2.2.4 WESTERN BLOT ANALYSIS 28
2.2.5 ANTIBODIES 28
2.3 PROTEIN EXPRESSION AND PURIFICATION FROM E. COLI 29
2.3.1 HETEROLOGOUS PROTEIN TEST EXPRESSION IN E. COLI
ROSETTA BLUE (DE3) AND BL21 29
2.3.2 BACTERIAL CELL LYSIS 29
2.3.3 DESCRIPTION OF INDIVIDUAL PROTEIN PURIFICATION STRATEGIES 29
2.3.3.1 PURIFICATION OF T. BRUCEI DOT1A AND DOT1B METHYLTRANSFERASES 29
2.3.3.2 PURIFICATION OF T. BRUCEI ASFLA AND ASFLB HISTONE CHAPERONES 30
2.3.3.3 PURIFICATION OF T. BRUCEI HISTONE PROTEINS FROM INCLUSION BODIES
31
2.4 NUCLEOSOME ASSEMBLY 32
2.4.1 HISTONE OCTAMER ASSEMBLY 32
2.4.2 SIZE EXCLUSION CHROMATOGRAPHY 32
2.4.3 HISTONE TETRAMER REFOLDING 33
2.4.4 NUCLEOSOME ASSEMBLY 33
2.4.5 LARGE-SCALE NUCLEOSOME ASSEMBLY 34
2.5 FUNCTIONAL ANALYSIS OF PURIFIED PROTEINS 34
2.5.1 HISTONE METHYLTRANSFERASE ASSAY 34
2.5.2 IN VITRO ASFL PULL-DOWN ASSAY 34
2.5.3 EX VIVO ASFL PULL-DOWN ASSAY 35
2.6 HOMOLOGY MODELING OF DOT1 METHYLTRANSFERASES 35
3 RESULTS 37
3.1 RECONSTITUTION OF TRYPANOSOMAL NUCLEOSOMES 37
3.1.1 EXPRESSION OF TRYPANOSOMAL HISTONE PROTEINS 37
3.1.2 NUCLEOSOME ASSEMBLY 39
3.2 ASFL HISTONE CHAPERONES 42
3.2.1 PURIFICATION OF HISTONE CHAPERONES ASFLA AND ASFLB 42
3.2.2 ASFLA BUT NOT ASFLB BINDS TO HISTONE H3/H4 DIMERS IN VITRO 43
3.3 RECOMBINANT EXPRESSION AND PURIFICATION OF DOT1
ENZYMES 44
3.4 DOT1
HISTONE METHYLTRANSFERASE ASSAY 45
3.5 DOT1A AND DOT1B ARE DISTRIBUTIVE ENZYMES IN VITRO 46
3.6 DOT1
HOMOLOGY MODELS 47
3.7 STRUCTURE-GUIDED MUTATIONS CHANGE THE PRODUCT-SPECIFICITY OF DOT1
ENZYMES 50
3.8 THE N-TERMINAL REGION CONTRIBUTES TO FORMATION OF THE LYSINE-BINDING
CHANNEL 53
4 DISCUSSION 57
4.1 ASFL HISTONE CHAPERONES 57
TABLE OF CONTENTS
4.1.1 THE TRYPANOSOMAL ASFLA AND ASFLB SYSTEM 57
4.1.2 CONCLUSION AND FUTURE PERSPECTIVE 58
4.2 DOT1
METHYLTRANSFERASES 58
4.2.1 THE MOLECULAR BASIS FOR PRODUCT-SPECIFICITY OF TRYPANOSOMAL DOT1
ENZYMES 58
4.2.2 CONTRIBUTION OF RESIDUES OUTSIDE OF THE CATALYTIC CORE 60
4.2.3 SUBSTRATE-TARGETING OF DOT1 ENZYMES 60
4.2.4 HISTONE UBIQUITYLATION 61
4.2.5 THE TRYPANOSOMAL DOT1A N-TERMINUS AS A POTENTIAL DRUG TARGET 62
4.2.6 CONCLUSION AND FUTURE PERSPECTIVE 62
5 REFERENCES 64
5 PUBLICATIONS 76
5 CURRICULUM VITAE 77
|
| any_adam_object | 1 |
| author | Dindar, Gülcin 1983- |
| author_GND | (DE-588)1059083337 |
| author_facet | Dindar, Gülcin 1983- |
| author_role | aut |
| author_sort | Dindar, Gülcin 1983- |
| author_variant | g d gd |
| building | Verbundindex |
| bvnumber | BV042217327 |
| collection | ebook |
| ctrlnum | (OCoLC)898031986 (DE-599)DNB1058765655 |
| discipline | Biologie Medizin |
| format | Thesis Book |
| fullrecord | <?xml version="1.0" encoding="UTF-8"?><collection xmlns="http://www.loc.gov/MARC21/slim"><record><leader>02145nam a2200481 c 4500</leader><controlfield tag="001">BV042217327</controlfield><controlfield tag="003">DE-604</controlfield><controlfield tag="005">20150113 </controlfield><controlfield tag="007">t</controlfield><controlfield tag="008">141201s2014 gw ad|| m||| 00||| eng d</controlfield><datafield tag="015" ind1=" " ind2=" "><subfield code="a">14,H11</subfield><subfield code="2">dnb</subfield></datafield><datafield tag="016" ind1="7" ind2=" "><subfield code="a">1058765655</subfield><subfield code="2">DE-101</subfield></datafield><datafield tag="035" ind1=" " ind2=" "><subfield code="a">(OCoLC)898031986</subfield></datafield><datafield tag="035" ind1=" " ind2=" "><subfield code="a">(DE-599)DNB1058765655</subfield></datafield><datafield tag="040" ind1=" " ind2=" "><subfield code="a">DE-604</subfield><subfield code="b">ger</subfield><subfield code="e">rakddb</subfield></datafield><datafield tag="041" ind1="0" ind2=" "><subfield code="a">eng</subfield></datafield><datafield tag="044" ind1=" " ind2=" "><subfield code="a">gw</subfield><subfield code="c">XA-DE</subfield></datafield><datafield tag="049" ind1=" " ind2=" "><subfield code="a">DE-12</subfield><subfield code="a">DE-384</subfield><subfield code="a">DE-473</subfield><subfield code="a">DE-703</subfield><subfield code="a">DE-1051</subfield><subfield code="a">DE-824</subfield><subfield code="a">DE-29</subfield><subfield code="a">DE-91</subfield><subfield code="a">DE-19</subfield><subfield code="a">DE-1049</subfield><subfield code="a">DE-92</subfield><subfield code="a">DE-739</subfield><subfield code="a">DE-898</subfield><subfield code="a">DE-355</subfield><subfield code="a">DE-706</subfield><subfield code="a">DE-20</subfield><subfield code="a">DE-1102</subfield></datafield><datafield tag="084" ind1=" " ind2=" "><subfield code="a">570</subfield><subfield code="2">sdnb</subfield></datafield><datafield tag="084" ind1=" " ind2=" "><subfield code="a">610</subfield><subfield code="2">sdnb</subfield></datafield><datafield tag="100" ind1="1" ind2=" "><subfield code="a">Dindar, Gülcin</subfield><subfield code="d">1983-</subfield><subfield code="e">Verfasser</subfield><subfield code="0">(DE-588)1059083337</subfield><subfield code="4">aut</subfield></datafield><datafield tag="245" ind1="1" ind2="0"><subfield code="a">Molecular basis for product-specificity of DOT1 methyltransferases in Trypanosoma brucei</subfield><subfield code="c">vorgelegt von Gülcin Dindar</subfield></datafield><datafield tag="246" ind1="1" ind2="3"><subfield code="a">Die molekularen Grundlagen der Produktspezifität von DOT1 Methyltransferasen in Trypanosoma brucei</subfield></datafield><datafield tag="264" ind1=" " ind2="1"><subfield code="c">2014</subfield></datafield><datafield tag="300" ind1=" " ind2=" "><subfield code="a">77 Bl.</subfield><subfield code="b">Ill., graph. Darst.</subfield><subfield code="c">30 cm</subfield></datafield><datafield tag="336" ind1=" " ind2=" "><subfield code="b">txt</subfield><subfield code="2">rdacontent</subfield></datafield><datafield tag="337" ind1=" " ind2=" "><subfield code="b">n</subfield><subfield code="2">rdamedia</subfield></datafield><datafield tag="338" ind1=" " ind2=" "><subfield code="b">nc</subfield><subfield code="2">rdacarrier</subfield></datafield><datafield tag="502" ind1=" " ind2=" "><subfield code="a">Würzburg, Univ., Diss., 2014</subfield></datafield><datafield tag="546" ind1=" " ind2=" "><subfield code="a">Zsfassung in dt. und engl. Sprache</subfield></datafield><datafield tag="650" ind1="0" ind2="7"><subfield code="a">Trypanosoma brucei</subfield><subfield code="0">(DE-588)4186350-1</subfield><subfield code="2">gnd</subfield><subfield code="9">rswk-swf</subfield></datafield><datafield tag="650" ind1="0" ind2="7"><subfield code="a">Chromatin</subfield><subfield code="0">(DE-588)4010152-6</subfield><subfield code="2">gnd</subfield><subfield code="9">rswk-swf</subfield></datafield><datafield tag="650" ind1="0" ind2="7"><subfield code="a">Histon-Methyltransferase</subfield><subfield code="0">(DE-588)4820080-3</subfield><subfield code="2">gnd</subfield><subfield code="9">rswk-swf</subfield></datafield><datafield tag="655" ind1=" " ind2="7"><subfield code="0">(DE-588)4113937-9</subfield><subfield code="a">Hochschulschrift</subfield><subfield code="2">gnd-content</subfield></datafield><datafield tag="689" ind1="0" ind2="0"><subfield code="a">Trypanosoma brucei</subfield><subfield code="0">(DE-588)4186350-1</subfield><subfield code="D">s</subfield></datafield><datafield tag="689" ind1="0" ind2="1"><subfield code="a">Histon-Methyltransferase</subfield><subfield code="0">(DE-588)4820080-3</subfield><subfield code="D">s</subfield></datafield><datafield tag="689" ind1="0" ind2="2"><subfield code="a">Chromatin</subfield><subfield code="0">(DE-588)4010152-6</subfield><subfield code="D">s</subfield></datafield><datafield tag="689" ind1="0" ind2=" "><subfield code="5">DE-604</subfield></datafield><datafield tag="776" ind1="0" ind2="8"><subfield code="i">Erscheint auch als</subfield><subfield code="n">Online-Ausgabe</subfield><subfield code="o">urn:nbn:de:bvb:20-opus-102524</subfield></datafield><datafield tag="856" ind1="4" ind2="1"><subfield code="u">https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-102524</subfield><subfield code="x">Resolving-System</subfield><subfield code="z">kostenfrei</subfield><subfield code="3">Volltext</subfield></datafield><datafield tag="856" ind1="4" ind2="2"><subfield code="m">DNB Datenaustausch</subfield><subfield code="q">application/pdf</subfield><subfield code="u">http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027655903&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA</subfield><subfield code="3">Inhaltsverzeichnis</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">ebook</subfield></datafield><datafield tag="999" ind1=" " ind2=" "><subfield code="a">oai:aleph.bib-bvb.de:BVB01-027655903</subfield></datafield></record></collection> |
| genre | (DE-588)4113937-9 Hochschulschrift gnd-content |
| genre_facet | Hochschulschrift |
| id | DE-604.BV042217327 |
| illustrated | Illustrated |
| indexdate | 2024-07-10T01:15:33Z |
| institution | BVB |
| language | English |
| oai_aleph_id | oai:aleph.bib-bvb.de:BVB01-027655903 |
| oclc_num | 898031986 |
| open_access_boolean | 1 |
| owner | DE-12 DE-384 DE-473 DE-BY-UBG DE-703 DE-1051 DE-824 DE-29 DE-91 DE-BY-TUM DE-19 DE-BY-UBM DE-1049 DE-92 DE-739 DE-898 DE-BY-UBR DE-355 DE-BY-UBR DE-706 DE-20 DE-1102 |
| owner_facet | DE-12 DE-384 DE-473 DE-BY-UBG DE-703 DE-1051 DE-824 DE-29 DE-91 DE-BY-TUM DE-19 DE-BY-UBM DE-1049 DE-92 DE-739 DE-898 DE-BY-UBR DE-355 DE-BY-UBR DE-706 DE-20 DE-1102 |
| physical | 77 Bl. Ill., graph. Darst. 30 cm |
| psigel | ebook |
| publishDate | 2014 |
| publishDateSearch | 2014 |
| publishDateSort | 2014 |
| record_format | marc |
| spelling | Dindar, Gülcin 1983- Verfasser (DE-588)1059083337 aut Molecular basis for product-specificity of DOT1 methyltransferases in Trypanosoma brucei vorgelegt von Gülcin Dindar Die molekularen Grundlagen der Produktspezifität von DOT1 Methyltransferasen in Trypanosoma brucei 2014 77 Bl. Ill., graph. Darst. 30 cm txt rdacontent n rdamedia nc rdacarrier Würzburg, Univ., Diss., 2014 Zsfassung in dt. und engl. Sprache Trypanosoma brucei (DE-588)4186350-1 gnd rswk-swf Chromatin (DE-588)4010152-6 gnd rswk-swf Histon-Methyltransferase (DE-588)4820080-3 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Trypanosoma brucei (DE-588)4186350-1 s Histon-Methyltransferase (DE-588)4820080-3 s Chromatin (DE-588)4010152-6 s DE-604 Erscheint auch als Online-Ausgabe urn:nbn:de:bvb:20-opus-102524 https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-102524 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=027655903&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Dindar, Gülcin 1983- Molecular basis for product-specificity of DOT1 methyltransferases in Trypanosoma brucei Trypanosoma brucei (DE-588)4186350-1 gnd Chromatin (DE-588)4010152-6 gnd Histon-Methyltransferase (DE-588)4820080-3 gnd |
| subject_GND | (DE-588)4186350-1 (DE-588)4010152-6 (DE-588)4820080-3 (DE-588)4113937-9 |
| title | Molecular basis for product-specificity of DOT1 methyltransferases in Trypanosoma brucei |
| title_alt | Die molekularen Grundlagen der Produktspezifität von DOT1 Methyltransferasen in Trypanosoma brucei |
| title_auth | Molecular basis for product-specificity of DOT1 methyltransferases in Trypanosoma brucei |
| title_exact_search | Molecular basis for product-specificity of DOT1 methyltransferases in Trypanosoma brucei |
| title_full | Molecular basis for product-specificity of DOT1 methyltransferases in Trypanosoma brucei vorgelegt von Gülcin Dindar |
| title_fullStr | Molecular basis for product-specificity of DOT1 methyltransferases in Trypanosoma brucei vorgelegt von Gülcin Dindar |
| title_full_unstemmed | Molecular basis for product-specificity of DOT1 methyltransferases in Trypanosoma brucei vorgelegt von Gülcin Dindar |
| title_short | Molecular basis for product-specificity of DOT1 methyltransferases in Trypanosoma brucei |
| title_sort | molecular basis for product specificity of dot1 methyltransferases in trypanosoma brucei |
| topic | Trypanosoma brucei (DE-588)4186350-1 gnd Chromatin (DE-588)4010152-6 gnd Histon-Methyltransferase (DE-588)4820080-3 gnd |
| topic_facet | Trypanosoma brucei Chromatin Histon-Methyltransferase Hochschulschrift |
| url | https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-102524 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027655903&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT dindargulcin molecularbasisforproductspecificityofdot1methyltransferasesintrypanosomabrucei AT dindargulcin diemolekularengrundlagenderproduktspezifitatvondot1methyltransferasenintrypanosomabrucei |