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    Investigating the interaction of HP1α with H1.4 in heterochromatin : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Genetics at Massey University, Palmerston North, New Zealand
    (Massey University, 2022) Smart, Maia Ellen Maxine
    Chromatin is a nucleoprotein complex which organises DNA within the cell and is essential for genome fidelity. However, the underlying mechanisms which regulate its formation are not fully understood. Chromatin forms through complex hierarchical folding. DNA wraps around octamers of the four core histones, H2A, H2B, H3 and H4, forming nucleosomes in an array called the 10 nm fibre, which is then condensed by linker histone H1 into the 30 nm fibre. RNA and architectural proteins such as Heterochromatin Protein 1α (HP1α) then fold the chromatin fibre into higher order domains, that ultimately partition the genome into domains of euchromatin and heterochromatin. The histone code model for HP1α induced heterochromatinization is through the chromodomain of HP1α binding to di- and tri-methylated lysine 9 of Histone 3 (H3K9me2/3). The H3K9me2/3 mark however, is present throughout the genome and so lacks the specificity for HP1α targeting. An interaction between HP1α and linker histone H1.4 has been identified previously, and through the establishment of an in vitro pulldown, it has been shown to be mediated by total RNA. To aid in building a model for HP1α targeting to telomeric heterochromatin, it was proposed that the RNA transcribed from this region, which associates with HP1α at the telomeres, could mediate this interaction. Therefore, Telomeric-repeat containing RNA (TERRA), for which HP1α has a high in vitro binding affinity, was tested to determine if it could mediate the interaction. TERRA was shown to mediate the interaction of HP1α with H1.4, indicating that this interaction could potentially be aiding in the targeting of HP1α to telomeric heterochromatin. Work to establish mononucleosomes to explore this interaction in a nucleosomal context has begun, however issues with unbound DNA and heterogeneous mononucleosome populations need to be overcome before these can be utilised. A model for HP1α targeting is proposed for reestablishment of telomeric heterochromatin after the S-phase of the cell cycle, through an interaction between H1.4 and HP1α, mediated by TERRA. These factors are essential in heterochromatin organisation, and their loss results in dysregulation which could lead to cancer and ageing.
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    Design, synthesis, and evaluation of cross-linked single-stranded DNAs as inhibitors of APOBEC3 enzymes : a thesis presented in partial fulfilment of the requirements of the degree of Doctor of Philosophy in Chemistry at Massey University, Manawatū, New Zealand
    (Massey University, 2021) Kurup, Harikrishnan Mohana
    Drug resistance is a major problem associated with anti-cancer chemo- and immunotherapies. Recent advances in the understanding of resistance mechanisms revealed that APOBEC3 (A3) family enzymes contribute to the development of drug resistance in multiple cancers. A3 enzymes are polynucleotide cytidine deaminases that convert cytosine to uracil (C→U) in single-stranded DNA (ssDNA) and in this way protect humans against viruses and mobile retro-elements. On the other hand, cancer cells use A3s, especially A3A and A3B, to mutate human DNA, and thus by increasing rates of evolution, cancer cells escape adaptive immune responses and resist drugs. However, as A3A and A3B are non-essential for primary metabolism, their inhibition opens a strategy to augment existing anticancer therapies and suppress cancer evolution. It is known that ssDNA bound to both A3A and to a chimeric A3B (A3BCTD) is not linear but adopts a distinctive U-shape, projecting the target cytosine into the active site. We hypothesized that locking DNA sequences into the observed U-shape may provide not only better substrates but also, appropriately modified, better inhibitors of A3 enzymes. To test our hypothesis that pre-shaped ssDNA mimicking the U-shape observed in ssDNA-A3 complexes can provide a better binder to A3 enzymes, a Cu(I)-catalyzed azide-alkyne cycloaddition was used to create a cross-link between two modified nucleobases in ssDNA. The resultant cytosine-containing substrate, where the cytosine sits at the apex of the loop, was deaminated faster by A3B than a standard, linear substrate. The cross-linked ssDNA was converted into an A3B inhibitor by replacing the 2'-deoxycytidine in the preferred TCA substrate motif by 2'-deoxyzebularine (dZ) or 5-fluoro-2'-deoxyzebularine (5-FdZ), a known inhibitor of single nucleoside cytidine deaminases. This strategy yielded the first nanomolar A3B inhibitor (Kᵢ = 100 ± 16 nM) and provides a platform for further development of modified ssDNAs as powerful A3 inhibitors. We also synthesized three seven-membered ring-containing nucleosides as transition-state analogues of cytosine deamination and incorporated them in a ssDNA sequence to test the inhibition of A3 enzymes. In this work we successfully synthesized a nucleoside with a seven-membered ring-containing double bond (ddiazep) and two nucleosides having a seven-membered ring with hydroxyl groups (R and S isomers) as a nucleobase. However, the inhibition of A3 by these compounds was not as good as by a ssDNA containing dZ. Interestingly, there was a difference in the inhibition produced by seven-membered ring-containing R and S isomers. The inhibition data showed that the relative S stereochemistry was essential in the inhibition of A3.