Dissertation submitted to the Combined Faculties for the Natural Sciences and for Mathematics of the Ruperto‐Carola University of Heidelberg, Germany for the degree of Doctor of Natural Sciences Presented by Master of Sciences Philipp Konstantin Zimmermann Born in Berlin‐Zehlendorf Oral examination: April 16th, 2015 Genome‐wide detection of induced DNA double strand breaks Referees: Prof. Dr. Christof von Kalle Prof. Dr. Stefan Wiemann I 1. INTRODUCTION 1 1.1 The DNA damage response and genomic instability 1 1.1.1 DNA damaging agents and the DNA damage response 1 1.2 DNA double‐strand break (DSB) repair pathways 3 1.2.1 The Non‐Homologous End Joining (NHEJ) Repair Pathway 4 1.2.2 Methods for the detection of DNA damage and DNA repair activity 6 1.3 Radio‐ and chemotherapy 7 1.3.1 Types of ionizing radiation 7 1.3.2 Radiation‐induced DNA damages 8 1.3.3 The topoisomerase family 9 1.3.4 Topoisomerase 2 catalytic cycle and targeting by anticancer drugs 10 1.3.5 Cancer therapy‐induced delayed genomic instability 11 1.4 Lentiviruses 11 1.4.1 History and phylogeny of lentiviruses 11 1.4.2 The lentiviral genome 12 1.4.3 The life cycle of lentiviruses 13 1.4.4 Structure of lentiviral vectors for gene therapy 15 1.5 Scientific aims 16 2. MATERIALS AND METHODS 18 2.1 Materials 18 2.1.1 Chemicals 18 2.1.2 Enzymes 19 2.1.3 Bacteria 19 2.1.4 Cell lines and primary cells 19 2.1.5 Antibodies 20 2.1.6 Plasmids 20 2.1.7 Oligonucleotides 20 2.1.7.1 Standard primers for q‐RT‐PCR 20 2.1.7.2 Primers used for linker cassettes in LAM‐PCR 21 2.1.7.3 Primers used for LAM‐PCR 21 2.1.7.4 Fusionprimer for Pyrosequencing 21 2.1.7.5 Primers and oligos used for direct DSB labeling approaches 22 2.1.8 Commercial kits 22 2.1.9 Buffers, Media, Solutions 23 2.1.10 Disposables 24 2.1.11 Equipment 24 2.1.12 Software and data bases 25 2.2 Methods 26 II 2.2.1 Cell Culture Methods 26 2.2.1.1 Cell Cultivation 26 2.2.1.2 Freezing and thawing of cells 26 2.2.1.3 Cell Counting 26 2.2.1.4 Transfection 27 2.2.1.5 Transduction 27 2.2.1.6 Virus production 28 2.2.1.7 Determining the lentiviral titer on Hela cells 28 2.2.1.8 MTT Assay 28 2.2.1.9 Immunostaining of H2AX foci 29 2.2.1.10 Inhibition of NHEJ‐repair activity by Nu7441 and Mirin 29 2.2.1.11 Irradiation of cells 30 2.2.1.12 Inhibition of Topoisomerase 2 in mammalian cells with doxorubicin and etoposide 30 2.2.1.13 Preparation for FACS 30 2.2.1.14 Synchronization of NHDF‐A in G1/G0, S and G2/M phase of the cell cycle 31 2.2.2 Molecular Biology Methods 32 2.2.2.1 Isolation of genomic DNA from cultivated cells 32 2.2.2.2 Determining the DNA concentration 32 2.2.2.3 Polymerase Chain Reaction (PCR) 32 2.2.2.4 DNA agarose electrophoresis 33 2.2.2.5 DNA isolation from agarose gel 33 2.2.2.6 Absolute quantitative real‐time PCR (q‐RT‐PCR) 33 2.2.2.7 Linear‐Amplification Mediated Polymerase Chain Reaction (LAM‐PCR) 34 2.2.2.8 Cleaning‐Up of PCR products using AMPure XP beads 39 2.2.2.9 Fusionprimer‐PCR 39 2.2.2.10 SureSelect Target Enrichment for Illumina Multiplexed Sequencing 40 2.2.2.11 Tdt‐mediated labeling of DSB sites 44 2.2.2.12 Biotin Quantification 47 2.2.2.13 Linker‐Amplification‐Mediated DSB Trapping (LAM‐DST) 48 2.2.2.14 Pyrophosphate sequencing 50 2.2.2.15 Cloning of PCR amplicons using the TOPO‐TA Cloning Kit 50 2.2.2.16 Transformation of circular DNA into chemically‐competent E.coli 50 2.2.2.17 Mini‐ and maxipreparation of plasmid DNA 51 2.2.2.18 Enzymatic DNA restriction digest 51 2.2.3 Bioinformatical Methods 51 2.2.3.1 Automated Sequence Analysis (HISAP) 51 2.2.3.2 A549 mRNA expression analysis 52 2.2.3.3 DNaseI Hypersensitive Sites 52 2.2.3.4 Histone modifications and Transcription Factor Binding Sites 52 2.2.3.5 Ingenuity Pathway Analysis (IPA) 53 2.2.3.6 Identification of DSB site clusters in the genome 53 3. RESULTS 54 3.1 IDLV‐mediated capturing of radiation‐induced DSB sites in vivo 54 III 3.1.1 Immunostaining of H2AX foci in irradiated cells 54 3.1.2 MTT assay to determine lethal dose values for etoposide and doxorubicin 55 3.1.3 IDLV‐Delivered DNA‐baits tag radiation‐induced and repaired DSB sites 56 3.1.4 IDLV DSB trapping in NHDF‐A with impaired NHEJ‐repair activity 58 3.1.5 Trapping and mapping of TOP2 poison‐induced DSB 58 3.1.6 Calculating the number of integrated IDLV copies per irradiated cell 59 3.1.7 SureSelect Target Enrichment for analyzing IDLV vector integrity 60 3.2 Analyzing early DSB repair events and kinetics of DSB induction at single nucleotide resolution 61 3.2.1 Genomic tagging of early‐repaired radiation‐induced DSB sites by IDLV 62 3.2.2 Synchronization of NHDF‐A and Hela cells in G1, S and G2 phase 62 3.2.3 Tdt‐mediated labeling of radiation‐induced DSB sites 63 3.2.4 Linker‐Amplification‐Mediated DSB‐Trapping for DSB labeling in real‐time 66 3.3 DSB site distribution in radiation‐surviving and expandable cell populations 68 3.3.1 Identification of radiation‐induced DSB sites by LAM‐PCR 68 3.3.2 DSB are not enriched on chromosomes, in genes and gene‐regulatory regions 69 3.3.3 IDLV integration at radiation‐induced DSB sites is mediated by NHEJ‐repair 71 3.4 Analyzing the influence of transcriptional activity, chromatin status and gene classes and networks on DSB site distribution 72 3.4.1 Trapping of Radiation‐Induced DSB is not influenced by transcriptional activity 72 3.4.2 The location of radiation‐induced DSB sites is composed of histone modifications defining active chromatin 73 3.4.3 Radiation‐induced DSB in radiation‐survivor cells are enriched in genes and networks regulating cell survival 77 3.4.4 Identification and analysis of radiation‐induced DSB sites over time 79 3.5 Identification of frequently damaged and repaired genomic regions 81 3.5.1 DSB Trapping in irradiated and passaged cells reveals common regions of radiation‐induced and repaired damage 81 3.5.2 Radiation‐related DSB hotspots overlap with genes involved in maintaining genome stability and DNA repair 84 3.5.3 DSB hotspots overlap with eu‐ to heterochromatin border regions 86 4. DISCUSSION 88 4.1 Immunostaining of H2AX is not suitable to detect DSB and genomic instabilities in cancer therapy surviving cell populations 88 4.2 NHEJ‐mediated IDLV integration at DSB sites stably marks DNA damage and repair sites in living cells 89 4.3 Identification of radiation‐induced DSB sites 90 4.3.1 Transcriptional activity before irradiation does not influence DSB site distribution 90 4.3.2 DSB site distribution is non‐random with respect to the genome accessibility 91 4.3.3 Genes involved in specific cellular processes are enriched for induced DSB 93 IV 4.3.4 Clonality of DSB site distribution over time 93 4.3.5 Radiation‐induced and repaired DSB sites cluster in hotspots in specific genomic regions 94 4.4 Methods for in situ labeling of induced DSB sites 96 5. SUPPLEMENT 98 5.1 Supplementary Figures 98 5.2 SupplementaryTables 110 5.3 Figure Index 135 5.3.1 Figures 135 5.3.2 Supplementary Figures 136 5.4 Table Index 137 5.4.1 Tables 137 5.4.2 Supplementary Tables 137 5.5 Zusammenfassung 139 5.6 Summary 140 5.7 Abbreviations 141 5.8 References 146 5.9 Publications and congress attendances 152 6. DANKSAGUNG 154 1 INTRODUCTION 1. INTRODUCTION 1.1 The DNA damage response and genomic instability 1.1.1 DNA damaging agents and the DNA damage response The DNA in the cell encodes the genetic information required for the functioning of all cells and living organisms. Every day, the DNA is under constant pressure to be destabilized by various processes and agents. These lesions can block DNA replication and transcription and are broadly divided according to their origin into either endogenous or exogenous (Figure 1) [1]. Endogenous processes such as DNA replication by DNA polymerases induce low levels of DNA damage. Other endogenous sources of DNA damage include genomic fragile sites, nucleases and reactive oxygen species (ROS) such as superoxide and hydrogen peroxide stemming from metabolic processes in the mitochondria [2]. These latter, highly reactive molecules form chemical bonds with other molecules within seconds of their production. Among their target molecules, the DNA is a susceptible target, in which ROS can cause base and sugar modifications, DNA‐protein crosslinks, single‐strand and double‐ strand breaks. In addition to endogenous processes, numerous exogenous sources also threaten the genomic integrity (Figure 1). The most pervasive form of exogenous DNA damaging agent is ultraviolet (UV) rays originating from sunlight that can hit the DNA directly and induce up to 100,000 DNA lesions per exposed cell [2].