Ribonucleotide excision repair (RER) is an RNase H2-dependent DNA-repair mechanism removing mis-incorporated ribonucleotides to maintain DNA stability. Decreased RNase H2 activity leads to an accumulation of ribonucleotides in the DNA, destabilizing and eventually damaging the DNA. This results in double-strand breaks, chromosome abberations, impaired segregation of defective chromosomes, and the formation of micronuclei with unstable nuclear membranes. Upon breakdown of the mironuclear envelope, the released chromatin triggers a cGAS-STING-dependent immune cascade that stimulates the production of type I interferons and cytokines. RNase H2 deficiency directly contributes to autoinflammation and autoimmunity and might further play a role in several types of cancer, aging and neurodegeneration. Therefore, RNase H2 activity is a promising diagnostic and prognostic marker. However, until today, no method for quantification of RNase H2 activity has been validated for a clinical use. Herein, a standard operating procedure for a high-throughput FRET-based whole cell lysate RNase H2 assay is implemented and validated delivering standard curves, statistical benchmarks and standardization to an externally validated control. Providing high sensitivity and strict linearity over a wide working range, the assay is applicable to various human cell or tissue samples with overall methodological assay variability from 8.6% to 16%. Human T cells were identified as a suitable cell type for the implementation of a clinical screening method, showing relatively small inter-individual variability when activity is normalized to cell number. Indeed, decreased RNase H2 activity was detected in T cells from one patient with systemic sclerosis and two patients with systemic lupus erythematosus who carried RNASEH2 mutations known to disrupt enzyme function in vitro compared with a control group of 24 healthy donors. With these findings, this dissertation provides fundamentals for the implementation of an RNase H2 assay screening method in the clinical setting. For the actual clinical application, however, the establishment of a significantly larger control group is necessary. This might allow identification of further inter-individual variables influencing RNase H2 activity and facilitate the determination of a threshold below which a reduction of RNase H2 activity is likely to become clinically relevant. Phenotypic effects of RNASEH2 mutations are often assessed in experiments with recombinant enzyme. However, this does not allow conclusions to be drawn about the extent to which the mutations affect enzyme activity through transcriptional, post-transcriptional, translational, or post-translational processes. In contrast, direct measurement of RNase H2 activity in cell lysates facilitates a more comprehensive assessment of the clinical relevance of genetic variants. To assess whether HeLa cell models are suitable for studying the intracellular effects of RNASEH2 mutations on enzyme activity, the well-studied RNASEH2B mutation A177T was inserted into HeLa cells using CRISPR/Cas9. However, due to high variability of RNase H2 activity in the HeLa cell clones after transfection and clonal selection, and low targeting efficiency, this approach has limited potential in HeLa cells. Direct use of immortalized cell lines derived from patient tissue, or a CRISPR/Cas approach in iPS cells might be more promising. As a secondary result, this study provides evidence that intracellular RNase H2 activity is increased in S, G2, and M phase of the cell cycle. In addition, increased RNase H2 activity was seen after stimulation with LPS and IL-2, and especially the mitogen PMA suggesting various pathways of RNase H2 activity regulation.:CONTENTS
CONTENTS 5
ABBREVIATIONS 8
ABSTRACT 13
ZUSAMMENFASSUNG 15
1 INTRODUCTION 17
1.1 RIBONUCLEOTIDE EXCISION REPAIR IN HEALTH AND DISEASE 17
1.1.1 Role and function of RER in mammals 17
1.1.2 Clinical relevance of dysfunctional RER 18
1.1.2.1 Aicardi-Goutières syndrome – a paradigm for autoimmunity 19
1.1.2.2 RNase H2 and malignancy 21
1.1.2.3 DNA-damage, aging and neurodegeneration 21
1.2 RIBONUCLEASE H2 21
1.2.1 Genetic and biochemical characteristics 21
1.2.2 Rnase H2 activity assays 23
1.2.3 Known mutations and their effects on enzymatic function 24
1.3 CRISPR/CAS: A TOOL FOR TARGETED MUTAGENESIS 27
1.4 AIM OF THIS THESIS 28
2 MATERIAL AND METHODS 29
2.1 MATERIAL 29
2.1.1 Chemicals and Reagents 29
2.2.1.1 Nucleic acids 29
2.2.1.2 Enzymes 30
2.2.1.3 Antibodies 31
2.2.1.4 Buffers and solutions 31
2.2.1.5 Cell growth media 32
2.2.1.6 Basic chemicals and reagents 32
2.1.2 Consumables 34
2.1.3 Kits 34
2.1.4 Devices 35
2.1.5 Cell lines 36
2.1.6 Animal clones 36
2.1.7 Software, databases and websites 37
2.2 METHODS 37
2.2.1 Cell methods 37
2.2.1.1 Isolation of primary cells 37
2.2.1.2 Cell culture 39
2.2.1.3 Cell analysis 41
2.2.2 Nucleic acid methods 42
2.2.2.1 DNA isolation via isopropanol precipitation 42
2.2.2.2 DNA isolation via ethanol precipitation in 96-well plates 42
2.2.2.3 DNA quantification 43
2.2.2.4 Primer design for PCR 43
2.2.2.5 Polymerase chain reaction (PCR) and gel electrophoresis 43
2.2.2.6 Purification of PCR products 44
2.2.2.7 Cloning methods 44
2.2.2.8 DNA Sequencing 45
2.2.3 Protein methods 46
2.2.3.1 Protein extraction from cells 46
2.2.3.2 Protein quantification 47
2.2.4 Statistics and informatics 47
2.2.5 CRISPR/Cas methods 47
2.2.5.1 sgRNA design 47
2.2.5.2 Cloning of plasmids containing sgRNA cassettes 48
2.2.5.3 Design of repair templates 49
2.2.5.4 Generating genetically modified HeLa cells using CRISPR/Cas9 49
2.2.5.5 Genotyping of cell clones generated by CRISPR/Cas9 50
2.2.6 RNase H2 activity 50
2.2.7 Control group inclusion criteria 50
3 RESULTS 51
3.1 ESTABLISHING THE RNASE H2 ASSAY 51
3.1.1 Method establishment 51
3.1.1.1 Methodological approach 51
3.1.1.2 Assay workflow and normalization 51
3.1.1.3 Establishing basic assay settings 54
3.1.1.4 Time-resolved measurement 54
3.1.1.5 Establishing controls 56
3.1.1.6 Fluorescence standard curves 62
3.1.1.7 Interpretation of the fluorescence progress curve 62
3.1.1.8 Steady-state kinetics: Definition of assay end-points 65
3.1.1.9 Standardization to externally validated controls 66
3.1.1.10 Ruggedness 68
3.1.1.11 Influence of cell cycle and stimulation on RNase H2 activity 70
3.1.2 Assay precision 70
3.1.2.1 Coefficient of variation 70
3.1.2.2 Experimental design 71
3.1.2.3 Error levels I – III: from linear regression to pipetting error 71
3.1.2.4 Error level IVa and IV: quantification error 75
3.1.2.5 Error levels V and VI: cell preparation errors 77
3.1.2.6 Calculation of individual CVs 79
3.1.2.7 Replication of individual assay steps and the effective CV 81
3.1.2.8 Inter-assay variability the use of standards 82
3.1.3 RNase H2 activity of different cell types 82
3.2 ESTABLISHING A SCREENING STRATEGY FOR RNASE H2 ACTIVITY 85
3.2.1 Choice of cell type and cell isolation 85
3.2.2 Recruitment of the control group 86
3.2.3 Biological variability of RNase H2 activity in B cells and T cells 86
3.2.4 Sample size and effect size 89
3.2.5 Reduced RNase H2 Activity in T Cells of Patients with Systemic Autoimmunity 91
3.3 GENERATION OF AN RNASEH2BA177T CELL MODEL 93
3.3.1 Experimental design 93
3.3.2 Genotyping results 94
3.3.3 Impact of the RNASEH2B A177T mutation on RNase H2 activity 95
4 DISCUSSION 98
4.1 RNASE H2 ASSAY 98
4.1.1 Qualitative validity 98
4.1.1.1 Assay end-points 98
4.1.1.2 Determination of RNase H2 activity from enzyme progress curves 100
4.1.1.3 Normalization 102
4.1.1.4 Validation and control of systematic errors 104
4.1.2 Quantitative considerations 107
4.1.2.1 Sensitivity, precision and replication 107
4.1.2.2 Applicability for high-throughput analysis 108
4.1.3 Perspective 108
4.2 RNASE H2 ACTIVITY SCREENING IN HUMAN CD3+ CELLS 109
4.3 CELL MODELS FOR PATHOGENIC RNASE H2 VARIANTS 112
4.4 RNASE H2 FUNCTION AND REGULATION 113
4.4.1 RNase H2 and transcription 113
4.4.2 RNase H2 kinetic parameters 115
4.4.3 RNase H2 activity during the cell cycle and induction by PMA 115
4.4.4 RNase H2 activity in different cell types 117
REFERENCES 119
APPENDIX 134
APP. 1: ASSAY SUBSTRATES 134
APP. 2: ANALYSIS OF ERROR SOURCES 134
Biological errors 134
Procedural errors 137
APP. 3: QUBITTM PROTEIN ASSAY PERFORMANCE CHARACTERISTICS 139
APP. 4: ‘ACCURACY’ AND RELATED TERMS 140
APP. 5: CHARACTERISTICS OF THE SYSTEMIC SCLEROSIS PATIENT SSC1 141
APP. 6: RNASE H2 SUBUNIT PROTEIN EXPRESSION IN DIFFERENT TISSUES 142
APP. 7: PARADIGM CALCULATION OF THE EFFECTIVE METHODOLOGICAL CV 143
APP. 8: GENOTYPING RESULTS OF CRISPR/CAS9-GENERATED HELA CLONES 144
APP. 9: RNASE H2 ASSAY STANDARD OPERATING PROCEDURE 146
SOP 1 cell preparation and lysis 146
SOP 1.1 Material and reagents 146
SOP 1.2 Assay planning 146
SOP 1.3 Prepare cell pellets 147
SOP 1.4 Lysis 147
SOP 2 Qubit™ protein assay 148
SOP 2.1 Material 148
SOP 2.1 Working procedure 148
SOP 3 RNase H2 assay 150
SOP 3.1 Material and reagents 150
SOP 3.2 Prepare a plate layout and a pipetting scheme 151
SOP 3.3 Prepare the reaction buffer and substrates 151
SOP 3.4 Prepare your lysate premix (volume B, 65 µl) 151
SOP 3.5 Prepare the photometer 152
SOP 3.6 Start the reaction by adding volume A (55 µl) to the reaction plate 152
SOP 3.9 Insert the plate, perform gain adjustment and start the test run 152
SOP 3.10 Data analysis 152
SOP 4 Figures and Charts 155
RNase H2 assay work flow 155
Assay substrates 156
Chart A. Corrected CVs of all error levels 157
Estimation of the effective CV for a planned experiment 158
Chart B: RNaseH2 assay working range for different cell types 159
Chart C: Approximate cell yield of biological material 159
Chart D: Plate layout 160
Chart E: Pipetting scheme 160
Pipetting work flow 161
Chart F: Fluorescence raw data table 161
Calculation of standard catalytic activity using standard curves 162
Inter-assay comparability 163
ACKNOWLEDGMENTS 164
DECLARATIONS 165 / Ribonukleotid-Exzisionsreparatur ist ein RNase-H2-abhängiger DNA-Reparaturmechanismus, der durch die Entfernung fälschlich eingebauter Ribonukleotide die Integrität und Stabilität der DNA erhält. Eine verminderte RNase-H2-Aktivität führt zu einer Anhäufung von Ribonukleotiden in der DNA, wodurch die DNA destabilisiert wird und schließlich Schaden nimmt. Das Resultat sind Doppelstrangbrüche, Chromosomenabberationen, eine gestörte Segregation der defekten Chromosomen und die Bildung von „Mikrokernen“ mit instabilen Kernmembranen. Bei Zerfall dieser Mikrokern-Hüllen löst das freiwerdende Chromatin eine cGAS-STING-abhängige Immunkaskade aus, welche die Bildung von Typ-I-Interferonen und Zytokinen stimuliert. Verminderte RNase-H2-Aktivität trägt dadurch direkt zur Entstehung von Autoinflammation und Autoimmunität bei und spielt wahrscheinlich auch als Malignitätsfaktor einiger Karzinome, sowie bei Alterungsprozessen und Neurodegeneration eine Rolle. Daher kann RNase-H2-Aktivität als ein vielversprechender diagnostischer und prognostischer Marker angesehen werden. Bisher etablierte Methoden zur Messung der RNase-H2-Enzymaktivität verfügen jedoch nicht über die Standardisierung und Validierung, welche für den klinischen Einsatz notwendig sind. Diese Dissertation implementiert eine Standardvorgehensweise, Standardkurven und statistische Kenngrößen für einen FRET-basierten RNase-H2-Assay. Der Assay ist für die Anwendung mit Zelllysaten validiert, und liefert standardisierte Ergebnisse. Durch eine hohe Sensitivität und eine strikte Linearität über einen großen Arbeitsbereich kann der Assay in vielen verschiedenen Zell- oder Gewebetypen angewendet werden. Die Gesamt-Variabilität beträgt dabei zwischen 8,6 % bis 16 %. Aufgrund einer relativ niedrigen inter-individuellen Schwankung der zellulären RNase-H2-Aktivität sind menschliche T-Zellen ein geeigneter Zelltyp für klinische Vergleichsstudien. So konnte in T-Zellen einer Patientin mit Systemischer Sklerose und zweier Patientinnen mit Systemischem Lupus Erythematodes, welche bekannte heterozygote RNASEH2 Mutationen aufwiesen, eine verminderte RNase-H2-Aktivität im Vergleich zu einer Kontrollgruppe mit gesunden Probanden gefunden werden. Diese Dissertation liefert die Grundlagen für die Implementierung eines RNase-H2-Assays als klinisches Diagnostikum. Für eine tatsächliche klinische Anwendung ist jedoch die Etablierung einer deutlich größeren Kontrollgruppe notwendig. Dadurch könnten einerseits weitere interindividuelle Einflussgrößen auf die RNase-H2-Aktivität identifiziert werden. Andererseits könnte dies die Festlegung eines Schwellenwerts ermöglichen, unter welchem sich eine Reduktion der RNase-H2-Aktivität wahrscheinlich klinisch manifestiert. Zur Beurteilung der phänotypischen Auswirkungen von RNASEH2 Mutationen werden häufig Experimente mit rekombinanter RNase-H2-durchgeführt. Dies lässt allerdings keine Aussagen darüber zu, inwiefern die Mutationen die Enzymaktivität durch transkriptionelle, post-transkriptionelle, translationale oder post-translationale Prozesse beeinflussen. Die direkte Messung der RNase-H2-Aktivität in Zelllysaten ermöglicht eine umfassendere Bewertung der klinischen Relevanz genetischer Varianten. Zur Einschätzung ob HeLa-Zell-Modelle geeignet dafür sind, die intrazellulären Auswirkungen von RNASEH2-Mutationen auf die Enzymaktivität zu untersuchen, wurde mittels CRISPR/Cas9 die vielseits publizierte RNASEH2B-Mutation A177T in HeLa-Zellen eingefügt. Jedoch fand sich nach der Transfektion und Zellklonierung eine sehr hohe Variabilität der RNase-H2-Aktivität zwischen den HeLa-Zellklonen. Aufgrund der zudem relativ niedrigen Targeting-Effizienz scheinen HeLa-Zellen für diese Fragestellung ein wenig geeigneter Zelltyp zu sein. Die direkte Verwendung von immortalisierten Zelllinien aus Patientengewebe, oder die Anwendung von CRISPR/Cas in iPS-Zellen könnten vielversprechender sein. Als Nebenbefund fand sich in dieser Dissertation eine erhöhte RNase-H2-Aktivität in der S-, G2- und M-Phase des Zellzyklus, sowie nach der Stimulation mit LPS und IL-2, sowie insbesondere dem Mitogen PMA. Dies liefert Hinweise zu möglichen intrazellulären Regulationswegen der RNase-H2-Aktivität, über welche bisher wenig bekannt ist.:CONTENTS
CONTENTS 5
ABBREVIATIONS 8
ABSTRACT 13
ZUSAMMENFASSUNG 15
1 INTRODUCTION 17
1.1 RIBONUCLEOTIDE EXCISION REPAIR IN HEALTH AND DISEASE 17
1.1.1 Role and function of RER in mammals 17
1.1.2 Clinical relevance of dysfunctional RER 18
1.1.2.1 Aicardi-Goutières syndrome – a paradigm for autoimmunity 19
1.1.2.2 RNase H2 and malignancy 21
1.1.2.3 DNA-damage, aging and neurodegeneration 21
1.2 RIBONUCLEASE H2 21
1.2.1 Genetic and biochemical characteristics 21
1.2.2 Rnase H2 activity assays 23
1.2.3 Known mutations and their effects on enzymatic function 24
1.3 CRISPR/CAS: A TOOL FOR TARGETED MUTAGENESIS 27
1.4 AIM OF THIS THESIS 28
2 MATERIAL AND METHODS 29
2.1 MATERIAL 29
2.1.1 Chemicals and Reagents 29
2.2.1.1 Nucleic acids 29
2.2.1.2 Enzymes 30
2.2.1.3 Antibodies 31
2.2.1.4 Buffers and solutions 31
2.2.1.5 Cell growth media 32
2.2.1.6 Basic chemicals and reagents 32
2.1.2 Consumables 34
2.1.3 Kits 34
2.1.4 Devices 35
2.1.5 Cell lines 36
2.1.6 Animal clones 36
2.1.7 Software, databases and websites 37
2.2 METHODS 37
2.2.1 Cell methods 37
2.2.1.1 Isolation of primary cells 37
2.2.1.2 Cell culture 39
2.2.1.3 Cell analysis 41
2.2.2 Nucleic acid methods 42
2.2.2.1 DNA isolation via isopropanol precipitation 42
2.2.2.2 DNA isolation via ethanol precipitation in 96-well plates 42
2.2.2.3 DNA quantification 43
2.2.2.4 Primer design for PCR 43
2.2.2.5 Polymerase chain reaction (PCR) and gel electrophoresis 43
2.2.2.6 Purification of PCR products 44
2.2.2.7 Cloning methods 44
2.2.2.8 DNA Sequencing 45
2.2.3 Protein methods 46
2.2.3.1 Protein extraction from cells 46
2.2.3.2 Protein quantification 47
2.2.4 Statistics and informatics 47
2.2.5 CRISPR/Cas methods 47
2.2.5.1 sgRNA design 47
2.2.5.2 Cloning of plasmids containing sgRNA cassettes 48
2.2.5.3 Design of repair templates 49
2.2.5.4 Generating genetically modified HeLa cells using CRISPR/Cas9 49
2.2.5.5 Genotyping of cell clones generated by CRISPR/Cas9 50
2.2.6 RNase H2 activity 50
2.2.7 Control group inclusion criteria 50
3 RESULTS 51
3.1 ESTABLISHING THE RNASE H2 ASSAY 51
3.1.1 Method establishment 51
3.1.1.1 Methodological approach 51
3.1.1.2 Assay workflow and normalization 51
3.1.1.3 Establishing basic assay settings 54
3.1.1.4 Time-resolved measurement 54
3.1.1.5 Establishing controls 56
3.1.1.6 Fluorescence standard curves 62
3.1.1.7 Interpretation of the fluorescence progress curve 62
3.1.1.8 Steady-state kinetics: Definition of assay end-points 65
3.1.1.9 Standardization to externally validated controls 66
3.1.1.10 Ruggedness 68
3.1.1.11 Influence of cell cycle and stimulation on RNase H2 activity 70
3.1.2 Assay precision 70
3.1.2.1 Coefficient of variation 70
3.1.2.2 Experimental design 71
3.1.2.3 Error levels I – III: from linear regression to pipetting error 71
3.1.2.4 Error level IVa and IV: quantification error 75
3.1.2.5 Error levels V and VI: cell preparation errors 77
3.1.2.6 Calculation of individual CVs 79
3.1.2.7 Replication of individual assay steps and the effective CV 81
3.1.2.8 Inter-assay variability the use of standards 82
3.1.3 RNase H2 activity of different cell types 82
3.2 ESTABLISHING A SCREENING STRATEGY FOR RNASE H2 ACTIVITY 85
3.2.1 Choice of cell type and cell isolation 85
3.2.2 Recruitment of the control group 86
3.2.3 Biological variability of RNase H2 activity in B cells and T cells 86
3.2.4 Sample size and effect size 89
3.2.5 Reduced RNase H2 Activity in T Cells of Patients with Systemic Autoimmunity 91
3.3 GENERATION OF AN RNASEH2BA177T CELL MODEL 93
3.3.1 Experimental design 93
3.3.2 Genotyping results 94
3.3.3 Impact of the RNASEH2B A177T mutation on RNase H2 activity 95
4 DISCUSSION 98
4.1 RNASE H2 ASSAY 98
4.1.1 Qualitative validity 98
4.1.1.1 Assay end-points 98
4.1.1.2 Determination of RNase H2 activity from enzyme progress curves 100
4.1.1.3 Normalization 102
4.1.1.4 Validation and control of systematic errors 104
4.1.2 Quantitative considerations 107
4.1.2.1 Sensitivity, precision and replication 107
4.1.2.2 Applicability for high-throughput analysis 108
4.1.3 Perspective 108
4.2 RNASE H2 ACTIVITY SCREENING IN HUMAN CD3+ CELLS 109
4.3 CELL MODELS FOR PATHOGENIC RNASE H2 VARIANTS 112
4.4 RNASE H2 FUNCTION AND REGULATION 113
4.4.1 RNase H2 and transcription 113
4.4.2 RNase H2 kinetic parameters 115
4.4.3 RNase H2 activity during the cell cycle and induction by PMA 115
4.4.4 RNase H2 activity in different cell types 117
REFERENCES 119
APPENDIX 134
APP. 1: ASSAY SUBSTRATES 134
APP. 2: ANALYSIS OF ERROR SOURCES 134
Biological errors 134
Procedural errors 137
APP. 3: QUBITTM PROTEIN ASSAY PERFORMANCE CHARACTERISTICS 139
APP. 4: ‘ACCURACY’ AND RELATED TERMS 140
APP. 5: CHARACTERISTICS OF THE SYSTEMIC SCLEROSIS PATIENT SSC1 141
APP. 6: RNASE H2 SUBUNIT PROTEIN EXPRESSION IN DIFFERENT TISSUES 142
APP. 7: PARADIGM CALCULATION OF THE EFFECTIVE METHODOLOGICAL CV 143
APP. 8: GENOTYPING RESULTS OF CRISPR/CAS9-GENERATED HELA CLONES 144
APP. 9: RNASE H2 ASSAY STANDARD OPERATING PROCEDURE 146
SOP 1 cell preparation and lysis 146
SOP 1.1 Material and reagents 146
SOP 1.2 Assay planning 146
SOP 1.3 Prepare cell pellets 147
SOP 1.4 Lysis 147
SOP 2 Qubit™ protein assay 148
SOP 2.1 Material 148
SOP 2.1 Working procedure 148
SOP 3 RNase H2 assay 150
SOP 3.1 Material and reagents 150
SOP 3.2 Prepare a plate layout and a pipetting scheme 151
SOP 3.3 Prepare the reaction buffer and substrates 151
SOP 3.4 Prepare your lysate premix (volume B, 65 µl) 151
SOP 3.5 Prepare the photometer 152
SOP 3.6 Start the reaction by adding volume A (55 µl) to the reaction plate 152
SOP 3.9 Insert the plate, perform gain adjustment and start the test run 152
SOP 3.10 Data analysis 152
SOP 4 Figures and Charts 155
RNase H2 assay work flow 155
Assay substrates 156
Chart A. Corrected CVs of all error levels 157
Estimation of the effective CV for a planned experiment 158
Chart B: RNaseH2 assay working range for different cell types 159
Chart C: Approximate cell yield of biological material 159
Chart D: Plate layout 160
Chart E: Pipetting scheme 160
Pipetting work flow 161
Chart F: Fluorescence raw data table 161
Calculation of standard catalytic activity using standard curves 162
Inter-assay comparability 163
ACKNOWLEDGMENTS 164
DECLARATIONS 165
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa:de:qucosa:90014 |
| Date | 20 February 2024 |
| Creators | Schulz, Marian Simon |
| Contributors | Roers, Axel, Rösen-Wolff, Angela, Technische Universität Dresden |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, doc-type:doctoralThesis, info:eu-repo/semantics/doctoralThesis, doc-type:Text |
| Rights | info:eu-repo/semantics/openAccess |
| Relation | 10.3390/jcm12041598 |
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