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Topoisomerase IIa human genomic binding sites
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Dr Emma Clark
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Topoisomerase (topo) is an essential nuclear enzyme that alters the topology of DNA by cutting and re-ligating double stranded DNA (dsDNA) via the formation of a transient ‘cleavable-complex’. This ensures the integrity of the DNA during many essential nuclear processes such as transcription, replication and segregation of daughter chromosomes. Thus, the cellular importance of this enzyme has made it a key chemotherapy target for many anti-tumour drugs such as etoposide, which stabilizes topo II cleavable complexes. Cellular processes then convert these complexes into lethal DNA lesions that ultimately cause cell death. However, the exact mechanisms and specifically whether the precise genomic location of a stabilised cleavable complex is a factor in the differential cytotoxicity of chemotherapy drugs are currently not well understood. This thesis describes for the first time the use of the Chromatin Immunoprecipitation (ChIP) assay to discover novel in vivo genomic sites (toposites) at which topo IIα associates in the presence and absence of etoposide. These sites were characterised by the cloning and sequencing of individual ChIP derived DNA sites and analysed using bioinformatic web-based programs to discern as much chromosomal information about the DNA site as possible. In addition, hybridisation of ChIP derived DNA against a human chromosome 22 microarray allowed direct analysis of etoposide mediated and non-drug mediated topo IIα sites across the whole of this chromosome. Sites are discussed in relation to the functions of the enzyme and a novel topo IIα in vivo binding motif derived from non-drug and etoposide mediated toposites is presented for the first time. Cleavable complex stabilisation by etoposide in relation to the cell cycle phase is also described. Using a Fluorescence Activated Cell Sorter (FACS) and also an elutriation system, K562 cells were physically separated into each distinct cell cycle phase. Greatest cleavable complex formation was found in the G2M phase in parallel with topo II protein expression. Finally, the AT DNA base binding compound Hoechst is used in topo cleavable complex inhibition studies to demonstrate that base specific regions of the genome are sensitive to drug stabilised topo cleavable complex formation. The implications for cytotoxicity in light of the above findings are thus discussed.
University of Newcastle upon Tyne
Newcastle upon Tyne
PhD thesis submitted to the Institute for Cell and Molecular Biosciences, Biomedical Sciences, University of Newcastle upon Tyne
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