Genetic and biochemical analysis of the synaptic complex of invertase Gin [Elektronische Ressource] / vorgelegt von Marina Baumann (geborene Dyachenko)

Baumann, Marina

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2004
• Nombre de lectures: 12
• Langue: English
• Poids de l'ouvrage: 4 Mo

Extrait

Genetic and biochemical analysis of the synaptic complex
of invertase Gin










Dissertation
zur Erlangung des Doktorgrades der Fakultät für Biologie
an der Ludwig-Maximilians Universität München






vorgelegt von
Marina Baumann
(geborene Dyachenko)

München, Juli 2004













1. Gutachter Prof. Dr. Dirk Eick
2. Gutachter Prof. Dr. Michael Boshart














Dissertation eingereicht am: 25.07.2004
Tag der mündlichen Prüfung: 1.04.2005 TABLE OF CONTENTS I

TABLE OF CONTENTS


ABBREVIATIONS

1. INTRODUCTION 1
1.1 Site-specific recombination 1
1.2 Serine recombinases: resolvases and invertases 4
1.3 The DNA inversion system of bacteriophage Mu 7
1.3.1 Structure of the Gin invertase protein 9
1.4 FIS protein, its structure and role in the inversion stimulation 12
1.4.1 FIS-Gin interactions in the invertasome 15
1.5 Models of the synaptic complex and mechanism of DNA recombination 17
1.6 Arrangement of Gin dimers in the catalytic tetramer: a preliminary model 19
1.7 Modular structure of the recombinases 23
1.7.1 The Gin-ISXc5 resolvase chimera 24
1.8 Goals 26

2. RESULTS 28
2.1 Screening for the Gin H106T activating FIS mutant 28
2.1.1 DNA binding and dimerisation properties of FIS S14P 30
2.1.2 Effect of the FIS S14P on DNA inversion in vitro 31
2.2 Intermolecular interactions in the Gin invertase catalytic tetramer 32
2.3 In vitro tetramerisation (gix-gix paring) assay 39
2.4 Characterisation of the role of the C-terminal DNA binding domain
in the synaptic complex formation using the chimeric protein generated
by fusion of Gin invertase and ISXc5 resolvase 46
2.4.1 Mutagenesis of the ISXc5G10 chimera 47
2.4.2 Cluster substitutions in the G10 chimera’s DNA-binding domain 48
2.4.3 Tetramerisation activity of the G10 chimera 50
2.4.4 Generation and testing of reciprocal chimeras 53
2.5 Interaction of the catalytic domains during tetramer formation 56
2.5.1 Characterisation of the DNA binding properties of the recombination proteins 56
2.5.1.1 Binding of the proteins to the gix/res sites of different lengths 56
2.5.1.2 Interactions with ISXc5 res subsite I 58TABLE OF CONTENTS II
2.5.1.3 Interactions with the gix site 60
2.5.2 Formation of heterodimers 62
2.5.3 Interaction between catalytic domains – the “beads experiment” 64

3. DISCUSSION 66
3.1 Gin-FIS communication 67
3.2 Gin surfaces involved in the interactions of dimers in tetramer:
mutations that affect the tetramer formation are situated in the catalytic
domain 68
3.3 Why is the ISXc5G10 chimera inversion deficient? 76
3.4 Outlook 80

4. MATERIALS AND METHODS 82
4.1 Chemicals 82
4.2 Enzymes, proteins and reagents 83
4.3 Molecular markers 84
4.3.1 DNA-length molecular standards for agarose gel electrophoresis 84
4.3.2 Protein molecular weight standards 84
4.4 General buffers and solutions 85
4.5 Media 86
4.6 Escherichia coli strains 87
4.7 Oligonucleotides 89
4.8 Plasmids 90
4.8.1 Plasmids used in the present work 90
4.8.2 Plasmids constructed in the present work 91
4.8.2.1 Description of the plasmids construction 92
4.9 Methods of microbiology and genetic 98
4.9.1 Determination of E. coli cell density 98
4.9.2 Cultivation of E. coli strains 99
4.9.3 Transformation of E. coli with plasmid DNA 99
4.9.3.1 CaCl transformation 992
4.9.3.2 Electrotransformation by electroporation 99
4.10 Methods of molecular biology 100
4.10.1 DNA manipulations 100TABLE OF CONTENTS III
4.10.1.2 Measurement of the DNA concentration 100
4.10.2 Preparation of the short DNA fragments 100
4.10.3 Hybridisation of complementary oligonucleotides 102
4.10.4 Radioactive labelling of the DNA fragments 102
4.10. Extraction of the DNA fragments from the polyacrylamide gel 103
4.10.6 PCR analysis 103
4.10.6.1 PCR mutagenesis 104
4.10.6.1.1 Introduction of site-specific mutations using QuikChange
XL system (Stratagene) 104
4.10.6.1.2 “Megaprimer” method 105
4.10.6.1.3 Random mutagenesis 106
4.10.6.1.3.1 Mutagenesis of the fis gene 108
4.10.6.1.3.2 Mutagenesis of the C-terminal part of the ISXc5G10 chimera 109
4.10.7 Sequence analysis 109
4.10.7.1 Automatic sequencing 109
4.11 In vivo recombination assays 110
4.11.1 In vivo inversion 110
4.11.1.1 Selection of the Gin H106T activating FIS mutant 111
4.11.1.2 Selection of the G10 chimera mutants with an inversion-proficient
phenotype 111
4.11.2 In vivo resolution 112
4.12vitro inversion assay 112
4.13 Biochemical methods 113
4.13.1 Preparations of protein extracts 113
4.13.1.1 Crude extract of FIS 113
4.13.1.2 Overproduction and purification of proteins with His-tags 114
4.13.2 Determination of protein concentrations 115
4.13.3 Protein gel electrophoresis 116
4.13.3.1 Denaturing SDS – polyacrylamide protein gel electrophoresis 116
4.13.3.1.1 Staining of PAA gels with Coomassie Brilliant Blue 117
4.13.3.2 Gel retardation assay 117
4.13.4 DNase I Protection Assay 118
4.13.5 Tetramerisation assay 119
4.13.6 Protein cross-linking 119TABLE OF CONTENTS IV
4.13.7 Transfer of proteins onto nitrocellulose membranes: semi-dry blotting
system 120
4.13.7.1 Staining membranes with Ponceau S 120
4.13.8 Immunodetection of proteins in Western blot using horseradish
peroxidase-conjugated antibodies 120
4.13.9 Beads-experiment 121

5. SUMMARY 123

6. REFERENCES 125

ACKNOWLEDGMENTS 135
ABBREVIATIONS V

ABBREVIATIONS


ad “till end volume” MGB minor groove binding (motif)
Ap ampicillin min minute
app. approximately ml milliliter
bp base pair mMmillimolarity
BSA bovine serum albumin ng nanogramme
C-terminus carboxy-terminus NMR nuclear magnetic resonance
Cm chloramphenicol N-terminus amino-terminus
DEB1,2:3,4-diepoxybutane OD optical density
DMSF dimethylsulfate ORF open reading frame
DMSO dimethylsulfoxide PAA polyacrylamide
DNase desoxyribonuclease PAGE ide gel
dNTP deoxynucleoside triphosphate electrophoresis
dsDNA double stranded DNA PCR polymerase chain reaction
DTT dithiothreitol PEG polyethylenglycol
E. coli Escherichia coli r resistance
EDTA ethylenediaminetetraacetic acid rpm rounds per minute
et al. and others RT room temperature
EtBr ethidiumbromide sec second
FIS factor for inversion stimulation SDS sodiumdodecylsulphate
Gin G inversion protein st-DNA single-stranded DNA
H Odouble-distillated water Tris Trihydroxymethylamino- 2 dd
HRP horseradish peroxidase methane
HTH helix-turn-helix (motif) Tc tetracycline
hr hour TEMED tetramethylendiamine
tyrT tyrosine tRNA gene IPTGisopropyl-1-thio- β-D-
U unit, enzyme activity galactopyranoside
UV ultraviolet light Kan kanamycin
V volt kb kilobase = 1000 bp
W watt kDa kilodalton
w/v weight per volume l liter
wt wild type lacZ β-galactosidase gene
X-Gal 5-bromo-4-chloro-3-indolyl- µ micro-
β-D-galactopyranoside M molarity INTRODUCTION 1

1. INTRODUCTION

1.1 Site-specific recombination

One of the most striking features of the genome is its ability to change. There are two main
processes that underlie this ability: mutation and rearrangement (recombination) of genetic
material. However, whilst mutation is predominantly a spontaneous event, genetic
recombination usually occurs under a strict control of many factors and often employs an
exact mechanism. Dependent on this mechanism three distinct types of recombination
systems can be discerned: homologous, site-specific (including transposition) and,
illegitimate recombination systems.
As the name suggests, site-specific recombination involves an interaction of specific DNA
sites. Recombination occurs by a precise exchange of DNA strands between these sites and
formation of new recombinant joints. Site-specific recombination is distinguished from
homologous recombination primarily by the mechanism of DNA recognition: in
homobination the recognition takes place between two homologous DNA
sequences and the search for complementarity is performed using a DNA-protein filament
(formed e.g. by the RecA protein). In site-specific recombination, the recombination
proteins (recombinases) themselves mediate recognition between the two distant DNA
sites. These proteins catalyse the DNA strand breakage and reunion without any
requirement for a high-energy cofactor. Since the site-specific recombination involves a
reciprocal crossover within two short sites of homologous sequence without any DNA
synthesis or degradation it is said to be conservative (Campbell, 1981).
Site-specific recombination systems are ubiquitous throughout eubacteria, prevalent in
archaea, but occur only rarely in eukaryotes. Complex eukaryotic genomes, however, can
be precisely manipulated by applying site-specific recombination s