Structural and Functional Characterization of Non-Homologous End Joining Factors

DNA double strand breaks represent the most toxic form of DNA damage. In mammals, non-homologous end-joining (NHEJ) is the primary DNA repair pathway for such damage, preventing both carcinogenesis and accelerated aging. Structural understanding of this repair pathway has received considerable attention, but has been significantly limited by the inability to obtain structures of higher order nucleoprotein complexes. A main obstacle in this respect has been difficulty in obtaining highly purified proteins, sufficient for structural determination. Improved protein expression and purification methods developed in this thesis permitted several NHEJ complexes to be selected for structural studies. Among these, Ku70-DNA and Ku70-DNA-PAXX yielded promising preliminary results. In depth optimization for crystal growth was performed and resulted in a full-length PAXX homodimer structure as well as low-resolution diffraction data for a novel Ku70-DNA complex. The PAXX structure confirmed prior suggestions that the C-terminal region of PAXX is highly disordered

RESOLUTION OF PROXIMAL OXIDATIVE BASE DAMAGE AND 3′-PHOSPHATE TERMINI FOR NONHOMOLOGOUS END JOINING OF FREE RADICAL-MEDIATED DNA DOUBLE-STRAND BREAKS

Clustered damage to DNA is a signature mark of radiation-induced damage, which involves damage to the nucleobases and/or DNA backbone. Double-strand breaks created by damaging agents are detrimental to cell survival leading to chromosomal translocations. Normal cells employ Non-homologous end-joining because of its faster kinetics, to suppress chromosomal translocations. However, the presence of complex DNA ends constitutes a significant challenge to NHEJ. Location of Thymine glycol (Tg) at DSB ends was a potential hindrance to end joining. The substrate with Tg at the third position (Tg3) from the DSB joined better than when present at the fifth position (Tg5). However, hNTH1 assay showed Tg5 to be a better substrate than Tg3 for BER, potentially explaining the increased Tg removal and decreased end joining of Tg5 in extracts. Nonetheless, there appeared to be no preference in the susceptibility of 5’-Tg substrates with Tg at the second and third positions from DSB ends. Polynucleotide kinase phosphatase is crucial in restoring the 3′ hydroxyl, and 5′ phosphate ends at strand breaks. No other enzyme is known to possess PNKP’s activity in mammalian cells at DSBs. Experiments done with PNKP knockout cells have shown some activity similar to PNKP, which appeared to be a part of NHEJ and was not pharmacologically inhibited by PNKP inhibitor. Additionally, core NHEJ factors XRCC4 and XLF influenced the activities of PNKP. Overall, these experiments suggest that Tg repair is dependent on the position from DSB and an alternative enzyme processes 3′- PO, and 5′-OH ends

Artemis over-expression and radiosensitivity in human cell lines

This thesis was submitted for the degree of Doctor of Philosophy and awarded by Brunel University.The cellular radiosensitivity of two fibroblast cell lines derived from a breast cancer patient that “over-reacted” to radiotherapy (84BR) and a patient with multiple independent tumours (175BR) was examined. Both patients had not been previously diagnosed with a mutation in any known DNA repair gene. The clonogenic assay revealed the 84BR and 175BR cell lines were hypersensitive to gamma radiation when compared to repair normal NB1 and 1BR.3 fibroblast cells. In addition, DNA DSB repair was found to be defective in both patient cell lines due to the abnormal persistence of -H2AX foci over a 24 hour time point in the nuclei of gamma irradiated cells when compared to normal fibroblasts. Also normal response to the cross-linking agent nitrogen mustard in a clonogenic assay was observed in 84BR and 175BR cell lines indicating a normal homologous recombination (HR) DNA repair pathway (since HR is essential for DNA crosslink repair). From these data it was concluded that these cells were defective in one or more components of the Non Homologous End Joining (NHEJ) pathway. The Artemis gene which has an endonuclease activity in the NHEJ repair pathway trims the ends of the double strand breaks before the two ends are ligated. Quantitative real time PCR analysis detected approximately 1.5 to 2 fold over-expression in Artemis gene in 84BR and 175BR cell lines compared to normal cells. Also an increase in the level of apoptosis before and following radiation exposure and a failure to efficiently repair DNA DSB were observed in the patient cell lines. Consequently, it was demonstrated in the cell lines described in this study that increased expression of Artemis endonuclease leads to abnormal and illegitimate DNA DSBs due to unregulated action of the protein thus, contributing to increased radiosensitivity and elevated apoptosis

Study of DNA repair and recombination mechanisms in Chinese hamster ovary cells

The CRISPR nuclease systems greatly facilitate targeted genome modifications in mammalian cells. The outcome of genome editing depends on the involved DNA double strand break (DSB) repair pathways. While the classical non-homologous end-joining and the poorly defined alternative end-joining (alt-EJ) DSB repair pathways can cause imprecise repair and thus gene inactivations, the homologous recombination (HR) pathway often introduces precise modifications. Although CRISPR is highly efficient at inactivating single genes, it is inefficient at introducing precise genome modifications. Moreover, its efficiency at inactivating multi-locus DNA sequences such as highly repetitive endogenous viral elements also remains limited. This thesis addressed these limitations by better characterizing DSB repair pathways in Chinese hamster ovary (CHO) cells - the most widely used production cell host for therapeutic proteins. In this thesis, I first aimed at identifying rate-limiting factors to improve HR-mediated genome editing. Second, I strove for studying approaches to inactivate repetitive endogenous retroviruses (ERV) presumably releasing viral particles into the CHO supernatant. To identify factors limiting HR, we established two chromosomal CHO assays that measure HR activity based on the correction of a GFP loss-of-function mutation. By using knockdown and overexpression studies, we found that efficient HR-mediated genome editing depended on certain alt-EJ activities. Furthermore, we observed that alt-EJ contribution to HR correlates with the nuclease type and the location of the DSB site relative to the GFP mutation. These observations suggest that alt-EJ and HR repair pathways tightly interact and challenges the common perception of alt-EJ opposing HR. Finally, among the tested repair factors, high Mre11 nuclease and Pari anti-recombinase as well as low Rad51 recombinase levels were the most rate-limiting factors for HR in CHO cells. Counteracting these bottlenecks improved HR efficiency by 75%. To inactivate repetitive ERVs, we transiently expressed a CRISPR-Cas9 nuclease that targets the gag gene of a specific transcriptionally active ERV group. Clones bearing a loss-of-function mutation in one particular ERV locus and corresponding mRNA produced considerably fewer particles loaded with viral RNA genomes. These findings indicated that a single ERV locus is responsible for the release of most, if not all, viral particles from CHO cells. Notably, ERV mutagenesis did not compromise cell growth, cell size or therapeutic protein production. In sum, this work provided novel strategies to improve HR-mediated genome editing and to inhibit viral particle release from CHO cells

Investigating the role of Tyrosyl-DNA Phosphodiesterase 1 in nuclear and mitochondrial DNA repair

Damages to the genetic materials arise throughout the lifespan of a cell, and elicit upregulation of DNA repair factors. Tyrosyl-DNA phosphodiesterase 1 (TDP1) is part of a DNA repair protein complex that specialises in the repair of DNA base modifications and single-strand breaks (SSBs). TDP1 removes a broad spectrum of chemical adducts from the 3’ end of a DNA strand break, including topoisomerase 1 (TOP1) peptide, during DNA transcription and replication. Inactivation or deletion of TDP1 is associated with cerebellar dysfunction and degeneration, with remarkably little extraneurological manifestation. The reason for the selective dependence of the cerebellar neurons on TDP1 activity is not clear. It was hypothesised that the TDP1 activity is upregulated in tissues with high levels of SSBs, either from DNA transcriptional activity, or reactive oxygen species (ROS)-induced damage. The aim of this doctoral project was therefore to identify and characterise the cellular mechanisms that regulate TDP1 activity. Our lab has previously shown that the Nterminus domain (NTD) of TDP1 covalently interacts with DNA ligase 3α. In this thesis, evidence has been presented to show that this interaction is regulated by the putative ATM/ATR/DNA-PK phosphorylation site, serine 81, to prolong TDP1 half-life, and enhance cellular survival after genotoxic stress. A second post-translational modification in the NTD by SUMOylation of the K111 residue was identified, enlightening a mechanism by which TDP1 is recruited to sites of transcription-mediated SSBs. To investigate the requirement for TDP1 in cells under high levels of oxidative stress, I have developed a mouse cellular model whereby the levels of endogenous ROS can be modulated by overexpression of the human anti-oxidant enzyme superoxide dismutase 1 (SOD1) or its toxic mutant SOD1G93A. Overexpression of SOD1G93A in Tdp1-/- MEFs induces accumulation of chromosomal SSBs and decreases survival after H2O2 challenge, while overexpression of SOD1 has a protective effect. Besides repair of ROS-induced TOP1-cc in the nucleus, TDP1 also repairs mitochondrial topoisomerase 1-mediated DNA breaks. This role is required during transcription and assembly of mitochondrial subunits of the electron transfer chain complexes, and has direct impact on mitochondrial respiration and ROS production. Collectively, these data provide mechanistic insights into regulation of TDP1-mediated chromosomal and mitochondrial DNA repair

Ride the Tide: Observing CRISPR/Cas9 genome editing by the numbers

Targeted genome editing has become a powerful genetic tool for modification of DNA sequences in their natural chromosomal context. CRISPR RNA-guided nucleases have recently emerged as an efficient targeted editing tool for multiple organisms. Hereby a double strand break is introduced at a targeted DNA site. During DNA repair genomic alterations are introduced which can change the function of the DNA code. However, our understanding of how CRISPR works is incomplete and it is still hard to predict the CRISPR activity at the precise target sites. The highly ordered structure of the eukaryotic genome may play a role in this. The organization of the genome is controlled by dynamic changes of DNA methylation, histone modification, histone variant incorporation and nucleosome remodelling. The influence of nuclear organization and chromatin structure on transcription is reasonably well known, but we are just beginning to understand its effect on genome editing by CRISP

Development and application of mass spectrometry based proteomics technologies to decipher ku70 functions

Master'sMASTER OF SCIENC

Telomere dysfunction in normal human epidermal keratinocytes

TRF2 is one of the main telomere binding proteins and a key regulator in protecting the telomere, the end of a linear chromosome. The telomere can be artificially "uncapped" by expression of myc-TRF2DeltaBDeltaM, a myc tagged dominant negative version of the protein. Both myc-TRF2 Full Length (FL) and myc-TRF2DeltaBDeltaM were retrovirally-infected into cells as part of an IRES-GFP construct. NHEK expressing GFP (and therefore either myc-TRF2 FL or myc-TRF2DeltaBDeltaM were selected at early time points using flow-sorting and this allowed either low or high expression levels to be isolated. Low or high levels of expression of myc-TRF2DeltaBDeltaM altered colony morphology, reduced clonogenicity and almost completely prevented proliferation of NHEK. Several markers of DNA damage were investigated and a small amount of p53- phosphoS15 was detected 2 days after expressing myc-TRF2DeltaBDeltaM in NHEK. The number of small colonies containing senescent cells was increased in NHEK expressing myc-TRF2DeltaBDeltaM compared with Vector only controls. Induction of DNA damage responses is undoubtedly a contributing factor and senescence at least one cellular outcome of uncapped telomeres in NHEK. Low levels of myc-TRF2 FL expression in NHEK also caused a reduction in cell proliferation, but not as severe as seen by expression of myc-TRF2DeltaBDeltaM. High levels of myc-TRF2 FL demonstrated an even greater reduction in proliferation, equivalent to myc-TRF2DeltaBDeltaM. Despite the reduced cell proliferation, NHEK expressing myc-TRF2 FL did not demonstrate any p53-phosphoS15. Excessive telomere processing may play a role in the dramatic effect seen in NHEK following expression of myc-TRF2 FL. The majority of human cancers are of epithelial derivation. As a major focus of cancer therapy is centered on exploiting the properties of the telomere, a full understanding of it's properties in an epithelial context is warranted. This thesis examines the effects of uncapping telomeres in Normal Human Epidermal Keratinocytes (NHEK), the first time an investigation into telomere status has taken place with a primary epithelial cell system

Investigating agricultural and biomedical applications of genome editors in large animals

Large animal species, such as cattle, sheep and pigs, have great potential value to scientific research. This is due to their physiological similarity to humans, meaning they make excellent disease models in addition to their inherent agricultural value. However, the efficiency with which such animals can be created has been a critical barrier to their use in bioscience. Research into creating genetically modified large animals has not progressed as rapidly as research on smaller mammals, such as mice, for two main reasons. Firstly, technologies such as pluripotent stem cells, which are well established in rodents, are lacking for large animals. Secondly, large animals cannot produce as many offspring within a given time frame as mice or rats. This, combined with the low efficiencies and lack of precision of current transgenic methods, severely reduces the likelihood of obtaining an animal with a desired genotype within a viable amount of time. Recently, new tools known as ’genome editors’ have been developed to facilitate genetic modification of animals. The vastly enhanced efficiency of these editors in comparison to previous gene targeting methods, combined with the fact that genome editors do not require marker genes to be used, mean that creating genetically modified livestock is now far more feasible. This thesis investigates whether two types of genome editor, TALENs and CRISPR/Cas9, can be used to produce genetically modified large animals for a range of applications. Genome editors were combined with interspecific blastocyst complementation techniques to produce chimeric rodents where the haematopoietic system is partially or fully derived from the donor cells. This work was carried out with a long-term aim of producing chimeric animals which could produce human organs suitable for transplantation. Initial blastocyst complementation experiments were carried out by injecting murine ESCs into wildtype rat blastocysts. One animal resulting from these injections showed chimerism in several tissues. Further experiments were carried out using rat ESCs and mouse blastocysts which were either Runx1-/- or Rag1-/-, however no additional chimeras were identified. In addition to these experiments, TALENs and sgRNAs were designed against Runx1 and Rag1 in sheep and pigs in order to create a large animal model for future blastocyst complementation experiments. Increasing animal productivity is a key step in meeting the demands of an increasing global population and tackling future food insecurities. TALENs and sgRNAs for use in the CRISPR/ Cas9 system were created to target the myostatin gene in sheep. Myostatin is a negative regulator of muscle growth and animals which acquire natural inactivating mutations in both myostatin alleles exhibit a well-characterised double-muscled phenotype, where total muscle mass is about 20% greater than that of a wildtype animal. Embryo microinjections were carried out using both types of genome editor and two edited lambs were produced, one from each editor. The TALEN-edited lamb was mosaic for a deletion of arginine 283 which, upon further analysis of the muscle, did not appear to cause a significant phenotype. The CRISPR-edited lamb was heterozygous for a 20bp deletion, causing the formation of a premature stop codon and severe truncation of the mature myostatin protein. Based on data from other myostatin-knockout animals, including the Belgian Blue cattle breed, this truncated protein is not thought to be functional. To determine if this is indeed the case, the CRISPR-edited lamb is now part of a breeding programme to amplify the edited allele. To discover if genome editors could be applied to create disease-resistant animals, the project focused on foot and mouth disease. Through a literature search and bioinformatic analysis of the bovine and porcine proteomes, three host genes which are cleaved by the virus were identified; eIF4A1, eIF4G1 and IKBKG. TALENs were designed to bind and cut at the FMDV protease cleavage sites in all three genes in order to disrupt protease cleavage and reduce viral replication by slowing viral disruption of the host translation and innate immune response pathways. Although none of the TALENs showed any signs of activity, this thesis sets out some potential directions for future work. In conclusion, this thesis shows that, despite some technical issues, genome editors are a promising technology for the creation of genetically modified livestock