University of Groningen
Single-molecule studies of the replisome
Spenkelink, Lisanne
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Publication date: 2018
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Spenkelink, L. (2018). Single-molecule studies of the replisome: Visualisation of protein dynamics in multi-protein complexes. Rijksuniversiteit Groningen.
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Single-molecule studies of the replisome
Visualisation of protein dynamics in multi-protein complexesPhD thesis
to obtain the degree of PhD of the University of Groningen
on the authority of the
Rector Magnificus Prof. Dr. E. Sterken and in accordance with
the decision by the College of Deans. and
to obtain the degree of PhD of the University of Wollongong
in the accordance with
the decision by the Graduate Research School Double PhD degree
This thesis will be defended in public on 21 September 2018 at 16.15 hours
by
Lisanne Maria Spenkelink
born on 19 May 1989 in Zwolle, the Netherlands
Promotor
Prof. dr. A.M. van Oijen
Copromotor Prof dr. B. Poolman Beoordelingscommissie Prof. dr. R. Fishel Prof. dr. W. Roos Prof. dr. D. Klostermeier Prof. dr. A. Oakley
Contents
1 Introduction 1
1.1 DNA replication . . . 1
1.2 Building complexity . . . 2
1.2.1 Bacteriophage T7 . . . 2
1.2.2 escherichia coli replisome . . . 3
1.2.3 Saccharomyces cerevisiae replisome . . . 6
1.3 Single-molecule techniques . . . 7
1.3.1 Why single molecules? . . . 7
1.3.2 Single-molecule fluorescence imaging . . . 9
1.3.3 Tethered-bead assay . . . 10
1.4 Scope of this thesis . . . 12
2 Watching cellular machinery in action, one molecule at a time. 13 2.1 Introduction . . . 14
2.2 Push, pull, poke and prod: Mechanical single-molecule techniques . . . 15
2.2.1 Atomic Force Microscopy . . . 17
2.2.2 Optical Tweezers . . . 19
2.2.3 Magnetic Tweezers . . . 21
2.3 What you see is what you get: Imaging techniques . . . 23
2.3.1 Total internal reflection fluorescence (TIRF) . . . 24
2.3.2 Local activation of dye (LADye), photoactivation, dif-fusion, and excitation (PhADE), point accumulation for imaging in nanoscale topography (PAINT) . . . . 26
2.3.3 Single-molecule fluorescence resonance energy trans-fer (smFRET) . . . 28
2.3.4 cryo-Electron Microscopy (cryo-EM) . . . 29
2.4 Two’s company, three’s a crowd: multi-protein complexes in crowded environments . . . 31
2.5 Outlook . . . 32
3 Single-molecule imaging at high fluorophore concentrations by Local Activation of Dye 35 3.1 Introduction . . . 36
3.2 Materials and methods . . . 38 iii
Contents
3.2.1 Dyes and proteins . . . 38
3.2.2 DNA construct . . . 39
3.2.3 Experimental setup . . . 40
3.2.4 Buffers for single-molecule measurements . . . 41
3.3 Results . . . 41
3.4 Discussion . . . 47
3.5 Supplementary information . . . 51
4 Quantification of ligand stoichiometries in liposomal drug de-livery systems using single-molecule fluorescence imaging 53 4.1 Introduction . . . 54
4.2 Results and discussion . . . 56
4.3 Conclusion . . . 63
4.4 Materials and Methods . . . 63
4.4.1 Labeling proteins with fluorophores. . . 63
4.4.2 Electrospray ionization mass spectrometry (ESI-MS). 64 4.4.3 Preparation of liposomes. . . 64
4.4.4 Intensity measurements for labeled proteins. . . 66
4.4.5 Measurement of protein density on liposomes. . . . 68
5 Single-molecule visualisation of fast polymerase turnover in the bacterial replisome 71 5.1 Introduction . . . 72
5.2 Results . . . 73
5.2.1 In vitro single-molecule observation of Pol III dy-namics . . . 73
5.2.2 Exchange of Pol III* complexes in vitro . . . 76
5.2.3 Quantification of exchange time of Pol III* in vitro . . 79
5.2.4 Exchange of Pol III* complexes in live cells . . . 81
5.3 Discussion . . . 83
5.4 Materials and Methods . . . 84
5.4.1 Protein expression and purification . . . 84
5.4.2 Expression plasmids . . . 85
5.4.3 Expression and purification of SNAP-alpha . . . 86
5.4.4 Fluorescent labeling of SNAP-alpha . . . 87
5.4.5 Ensemble strand-displacement DNA replication as-says . . . 88
Contents
5.4.6 Ensemble leading and lagging strand DNA
replica-tion assays . . . 88
5.4.7 In vitro single-molecule rolling-circle DNA replica-tion assay . . . 91
5.4.8 Measurement of the stoichiometry of Pol III*s at the replisome. . . 94
5.4.9 Fluorescent chromosomal fusions. . . 96
5.4.10 Growth rates of fluorescent chromosomal fusions. . 96
5.4.11 In vivo single-molecule visualization assays. . . 97
5.5 Supplementary figures . . . 100
6 Single-molecule visualization of SSB dynamics shows a com-petition between an internal-transfer mechanism and external exchange. 103 6.1 Introduction . . . 104
6.2 Results . . . 108
6.2.1 Vizualisation of SSB in vitro . . . 108
6.2.2 Dynamic behaviour of SSB in vitro . . . 111
6.2.3 SSB is recycled for many Okazaki fragments . . . . 113
6.2.4 Dynamic behavior of SSB in vivo . . . 117
6.3 Discussion . . . 120
6.4 STAR Methods . . . 123
6.4.1 Experimental model and subject details . . . 123
6.4.2 Method details . . . 123
7 The RarA protein of Escherichia coli creates DNA gaps be-hind the replisome 133 7.1 Introduction . . . 134
7.2 Results . . . 136
7.2.1 Rationale and outline . . . 136
7.2.2 RarA in vitro: RarA action creates gaps during DNA polymerase III-mediated DNA synthesis . . . 136
7.2.3 RarA in vivo . . . 141
7.2.4 RarA in vivo: (a) Effects of rarA deletions on cell growth. . . 143
7.2.5 RarA in vivo: (b) A rarA deletion suppresses the UV sensitivity of recF and recO mutations. . . 147
Contents
7.2.6 RarA in vivo: (c) A rarA deletion suppresses the DNA damage sensitivity of TLS polymerase mutants. 149 7.2.7 RarA in vivo: (d) A rarA deletion partially suppresses
the DNA damage sensitivity of a uvrA deletion mutant.152
7.3 Discussion . . . 153
7.3.1 Why do cells maintain a gap creating activity? . . . . 155
7.3.2 What is the trigger for gap formation? . . . 156
7.3.3 Promotion of lagging-strand gap creation . . . 156
7.3.4 What is the mechanism of polymerase detachment? 158 7.3.5 Implications of gap creation for TLS . . . 159
7.4 Materials and methods . . . 160
7.4.1 Replication proteins . . . 160
7.4.2 Labeling of beta with AF647 . . . 160
7.4.3 In vitro single-molecule rolling-circle DNA replica-tion assay . . . 161
7.4.4 Fluorescence polarization assay . . . 165
7.4.5 Reagents and growth conditions . . . 167
7.4.6 Strain construction . . . 167
7.4.7 Growth curves — plate reader . . . 167
7.4.8 Growth curves — spectrophotometer . . . 168
7.4.9 Growth competition assays . . . 168
7.4.10 Single-molecule time-lapse imaging and analysis . . 168
7.4.11 Single-molecule fluorescence imaging of cells grown in shaking culture . . . 170
7.4.12 Flow cytometry . . . 171
7.4.13 Spot plate drug/UV sensitivity assays . . . 173
7.5 Supplementary figures . . . 174
8 Single-molecule visualization of leading-strand synthesis by S. Cerevisiae reveals dynamic interaction of MTC with the replisome 179 8.1 Introduction . . . 180
8.2 Results . . . 182
8.2.1 Single-molecule visualization of leading-strand syn-thesis. . . 182
Contents
8.2.2 Single-molecule replication rates of pol epsilon
de-pendent leading strand synthesis. . . 185
8.2.3 Mcm10 increases the number of productive replica-tion events. . . 188
8.2.4 Addition of MTC increases replication rates of Pol epsilon dependent leading-strand synthesis. . . 189
8.2.5 MTC induces multiple rate changes within a single leading-strand replication complex. . . 190
8.2.6 MTC is transiently associated to the CMGE leading-strand replication fork complex. . . 193
8.3 Discussion . . . 194
8.4 Materials and Methods . . . 198
8.4.1 Protein expression and purification. . . 198
8.4.2 Linear fork DNA substrate . . . 200
8.4.3 Single-molecule tethered-bead assay . . . 201
8.4.4 Bead selection and processing . . . 202
8.4.5 Efficiency of leading-strand synthesis . . . 205
8.4.6 Ensemble leading-strand replication assays . . . 205
8.4.7 Code availability . . . 207
8.5 Supplementary figures . . . 208
9 Discussion 211 9.1 Improving single-molecule techniques . . . 211
9.2 Multi-site exchange mechanisms . . . 212
9.3 Replication and repair . . . 216
9.4 A more complex replisome . . . 217
10 Nederlandse Samenvatting 221
11 List of publications 225
12 Acknowledgements 227
