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Investigation of Dna Damage Response at Interstitial Telomeric Sites in Chinese Hamster Cell Lines

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Investigation of Dna Damage Response at Interstitial Telomeric Sites in Chinese Hamster Cell Lines INVESTIGATION OF DNA DAMAGE RESPONSE AT INTERSTITIAL TELOMERIC SITES IN CHINESE HAMSTER CELL LINES A thesis submitted for a degree of Doctor of Philosophy by Sheila Matta Castillo College of Health and Life Sciences Department of Life Sciences Brunel University July 2018 Declaration I hereby declare that the research presented in this thesis is my own work, except where otherwise specified, and has not been submitted for any other degree. Sheila Matta July 2018 2 Abstract Telomeres are highly specialised structures that protect the ends of linear chromosomes from damage. Telomeric DNA consists of tandem repeats of a specific sequence (TTAGGG)n that varies in length depending on the species. Two telomere maintenance mechanisms are known to exist. Telomerase, an enzyme that synthesises new tandem repeats onto the end of linear chromosome and ALT (Alternative Lengthening of Chromosomes), which uses recombination to lengthen telomeric sequences. In some mammalian species, both mechanisms are known to co-exist. Telomeric DNA that is found at non-telomere sites are referred to as interstitial telomeric sequences (ITS). Several studies have shown that ITS sites in the Chinese hamster are preferred sites for radiation and spontaneous chromosomal damage. Once damage occurs, repair mechanisms such as HR (homologous recombination) and NHEJ (non-homologous end joined) are activated. However, the contribution to this repair by telomerase and ALT is unknown. The aim of this work therefore is to investigate the contribution that above mention mechanisms (HR, NHEJ, telomerase and ALT) make to ITS repair in Chinese hamster cells. Classical cytogenetic techniques such as Giemsa staining, chromosomal aberration analysis, FISH (Fluorescence in situ hybridazation, IHC (Immunohistochemistry), TIF (Telomere dysfunction induced foci), were used to quantify chromosomal damage after IR (ionising radiation) treatment of normal and repair deficient (HR and NHEJ) Chinese 3 hamster cell lines. Molecular analysis employed the TRAP assay, and C-circle analysis to look at telomerase expression and ALT activity. Finally, siRNA was used to look at two genes which are important in the telomere maintenance pathways, ATRX and HDAC9, were knocked down using siRNA and cells were analysed using Western blotting and qPCR for the effects this had on telomerase and ALT. Our results show that chromatid breaks within ITSs sites were higher than expected in repair deficient cell lines. In addition, repair deficient cells recover more slowly to IR chromosomal damage than control cells, suggesting that HR and NHEJ play a part in the repair of ITS in Chinese hamster cells. siRNA knockdown revealed that ATRX and HDAC9 affected the expression of ALT and telomerase respectively in our cell lines. Taken together, our work provides evidence that show for the first time the co-existence of both telomerase and ALT activity in Chinese hamster cells. 4 TABLE OF CONTENTS Abstract …………………………………………………………………………………………………………………………….… 3 Table of Contents ……………………………………………………………………………………………………………..... 5 List of Figures ……………………………………………………………………………………………………..…….………… 8 List of Tablets ………………………………………………………………………………………………………………….….. 9 Acknowledgements ……………………………………………………………………………………………………………. 10 Abbreviations ……………………………………………………………………………………………………………………. 11 1. General Introduction …………………………………………………………………………………………….………....……… 15 1.1 Telomeres: Caps at the linear ends ……………………………………………………………………………….…............ 15 1.2 Telomere structure ………………………………………………………………………………………………………………..…... 16 1.3 G-quadruplex structure ……………………………………………………………………………………………………............ 17 1.4 Telomere function ………………………………………………………………………………………………………………………. 18 1.4.1 Telomere and Shelterin complex…………………………………………………………………..…………………………. 19 1.4.2 The end replication problem ……….…………………………………………………………………………….…............. 23 1.5 Telomerase ......................................................................................................................................... 24 1.5.1 Telomerase structure and function ................................................................................... 25 1.5.2 hTERT ........................................................................…………………………………………………….. 25 1.5.3 hTERC .................................................................................…………………………….……………… 27 1.6 Telomerase and cancer ...................................................................................................................... 27 1.7 Ectopic expression of telomerase in human cells ............................................................................. 28 1.8 Alternative lengthening of telomeres (ALT) ...................................................................................... 29 1.8.1 Alternative lengthening of telomeres hallmark …………………………………………………………… 30 1.9 Ionising radiation induced DNA damage ........................................................................................... 35 1.10 Chromosomal aberrations ............................................................................................................... 37 1.11 DNA damage response pathways activation ................................................................................... 37 1.12 Telomeres and interstitial telomere sites ........................................................................................ 41 1.12.1 Characterization of ITSs …………………………………………………………………………………..………… 42 1.12.2 Telomere binding protein and ITSs ................................................................................. 45 1.12.3 Radiation induced instability at ITSs ............................................................................................. 46 1.14 AIMS and Objectives ........................................................................................................................ 48 2. Materials and Methods …………………………………………………………………………………………….…..………… 49 2.1 Cell lines and tissue culture ............................................................................................................... 50 2.1.1 Chinese hamster cell lines.................................................................................................. 50 2.1.2 Human cancer cell lines ..................................................................................................... 52 2.2 Tissue culture procedure ................................................................................................................... 53 2.2.1 Cryopreservation of cells ................................................................................................... 54 2.2.2 Thawing of cryopreserved cells ......................................................................................... 54 2.2.3 Cell counting ...................................................................................................................... 54 2.2.4 Irradiation of cells .............................................................................................................. 55 2.3 Cytogenetic Analysis .......................................................................................................................... 55 2.3.1 Metaphase preparation in Chinese hamster cells ............................................................ 55 5 2.3.2 Giemsa staining ................................................................................................................. 56 2.3.3 Chromosomal aberration analysis ..................................................................................... 56 2.3.4 Metaphase Fluorescence in situ Hybridization ................................................................. 57 2.3.5 Hybridisation ...................................................................................................................... 58 2.3.6 Post-hybridisation washes ................................................................................................. 58 2.3.7 Image Analysis ................................................................................................................... 58 2.3.8 Analysis of the ITSs in percentage ..................................................................................... 59 2.4 Immunocytochemistry ....................................................................................................................... 60 2.4.1 Immunofluorescence detection of γ-H2AX foci ............................................................... 60 2.4.2 Telomere dysfunction-induced foci (TIF) assay ................................................................ 61 2.4.3 Immunofluorescence detection of ATRX foci ................................................................... 62 2.5 Quantification of Telomere Repeat Amplification (TRAP) protocol assay ........................................ 62 2.5.1 Protein concentration ........................................................................................................ 63 2.5.2 TRAP PCR reaction set up .................................................................................................. 64 2.6 Alternative lengthening of telomeres (ALT) c-circle assay ................................................................ 67 2.6.1 Genomic DNA extraction ................................................................................................... 67 2.6.2 DNA quantification ...........................................................................................................
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