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Analysis of the Architecture and Function of the Nuclear DNA Replication Apparatus in Trypanosoma Brucei

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Analysis of the Architecture and Function of the Nuclear DNA Replication Apparatus in Trypanosoma Brucei Tiengwe, Calvin (2010) Analysis of the architecture and function of the nuclear DNA replication apparatus in Trypanosoma brucei. PhD thesis. http://theses.gla.ac.uk/2307/ Copyright and moral rights for this thesis are retained by the author A copy can be downloaded for personal non-commercial research or study, without prior permission or charge This thesis cannot be reproduced or quoted extensively from without first obtaining permission in writing from the Author The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the Author When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given Glasgow Theses Service http://theses.gla.ac.uk/ [email protected] Analysis of the architecture and function of the nuclear DNA replication apparatus in Trypanosoma brucei Calvin Tiengwe BSc, MSc, MRes Submitted in fulfilment of the requirements for the Degree of Doctor of Philosophy Wellcome Centre for Molecular Parasitology and Institute of Infection, Immunity and Inflammation College of Medical Veterinary and Life Sciences, University of Glasgow October, 2010 ii Abstract DNA replication is central to the propagation of life and initiates by the designation of genome sequences as origins, where synthesis of a copy of the genetic material begins once per cell division. Despite considerable progress in understanding mitochondrial (kinetoplast) DNA replication in kinetoplastid parasites, little is known about nuclear DNA replication. The mechanism and machinery of DNA replication initiation is well-conserved among characterised eukaryotes. The six protein origin recognition complex (ORC, Orc1-Orc6), Cdc6, and Cdt1 are recruited sequentially to DNA, and once bound, they load the replicative helicase (MCM, a heterohexamer; subunits Mcm2-7) to form a pre-replicative complex (pre-RC) at potential origins of replication. The largest subunit of ORC, Orc1, is related in sequence to Cdc6, indicative of derivation from a common ancestor. Such an ancestral molecule appears still to function in archaea. These prokaryotes lack Cdc6 and possess a protein named Orc1/Cdc6, which appears to provide all ORC functions, since orthologues of Orcs2-6 are absent. In addition to this, archaeal orthologues of Cdt1 have not been clearly described, though potentially related factor, named WhiP (winged helix initiator protein), has been found. Comparative genome analysis of Trypanosoma brucei and related trypanosomatids (Leishmania major and Trypanosoma cruzi) revealed, remarkably, only a single ORC protein that is equally related to eukaryotic Orc1 and Cdc6 (named here TbORC1/CDC6). In addition, no clear homologue of Cdt1 was found. These observations have been interpreted as suggesting that origin designation in trypanosomatids, although eukaryotic, may be archaeal-like, raising numerous mechanistic and evolutionary questions. To test this hypothesis, and to dissect the process of nuclear DNA replication, a number of experiments are described in this thesis. We used RNA interference (RNAi) to demonstrate that knockdown of TbORC1/CDC6 in procyclic form (PCF) T. brucei cells inhibits nuclear DNA synthesis, as revealed by cell cycle analysis and a BrdU incorporation assay. iii Immunofluorescence and GFP-tagging showed that in procyclic form (PCF) cells TbORC1/CDC6 is a nuclear protein. In PCF cells, based on the evidence gathered, we confirm that TbORC1/CDC6 acts in nuclear DNA synthesis. In contrast, RNAi knockdown of TbORC1/CDC6 in bloodstream form (BSF) T. brucei cells resulted in the rapid accumulation of cells with more than two nuclei and two kinetoplasts, indicating a deregulation of the cell cycle, which is then followed by cell rapid cell death. This RNAi result provides greater evidence that TbORC1/CDC6 provides an essential function in the parasite, since RNAi depletion of TbORC1/CDC6 in PCF cells has a less pronounced effect on growth. Nevertheless, attempts to generate TbORC1/CDC6 null mutants failed in PCF cells, consistent with an essential role in this life cycle stage also. To study the molecular interactors of TbORC1/CDC6, we performed immunoprecipitation analyses. From this, we have identified one protein (gene ID, Tb927.10.13380) that acts as a component of the T. brucei pre- replicative machinery, and suggest that this is a previously unidentified orthologue of Orc4. We also indentified a further protein (gene ID, Tb927.10.7980) that may also act in T. brucei DNA replication, but whose identity and function are unclear. TbORC1/CDC6 appears not to interact directly with the TbMCM helicase (for which orthologues of all subunits can be identified), consistent with previous observations from a number of eukaryotic organisms, and contrary to reports in some archaeal species. MCM subunits in T. brucei form at least one subcomplex (TbMCM2/4/6/7) homologous to that previously observed for human, yeast, Drosophila, Xenopus and mouse MCM proteins. Taken together, these data appears to refute the hypothesis that the DNA replication pre-RC machinery in T. brucei is analogous to archaea. Rather, we propose that TbORC contains at least two components, TbORC1/CDC6 and Tb927.10.13380, more analogous to the eukaryotic model, suggesting that origin designation is not carried out by a single protein. To identify potential replication origin sequences, we performed chromatin immunoprecipitation with functional, epitope-tagged TbORC1/CDC6 in PCF iv cells and, using a high-resolution tiling array (NimbleGen) for T. brucei, we have mapped TbORC1/CDC6 binding sites along all the megabase chromosomes in the genome. Analyses of chromosomes 1-10 showed that 278 binding sites are sparsely located within the core of chromosomes, of which 114 loci (40%) co-localise with probable RNA Polymerase II transcription start sites, perhaps consistent with an origin function. In addition, a further 330 binding sites are present as high density clusters in subtelomeric VSG arrays, and 81 binding sites are associated with sub- telomeric elements, perhaps consistent with a non-origin function. Consistent with these results, RNAi knockdown of TbORC1/CDC6 led to derepression of metacyclic Variant Surface Glycoprotein (VSG) genes, suggesting that TbORC1/CDC6 plays a role in the epigenetic silencing at VSG expression sites in PCF T. brucei. Similar analysis of VSG expression in BSF cells, and of BSF VSGs in PCF cells, was less conclusive, perhaps suggesting differential functions of TbORC1/CDC6 in different life cycle stages or at different VSG expression sites. These analyses shed new light on the architecture and potential function of TbORC1/CDC6 in T. brucei nuclear DNA replication in general, as well as a potential association between replication and antigenic variation in T. brucei. v Table of Contents Abstract ................................................................................. ii Table of Contents ..................................................................... v List of Tables ........................................................................... x List of Figures......................................................................... xi List of Appendices .................................................................. xiv Acknowledgements ................................................................. xvi Author's declaration ............................................................... xvii List of Abbreviations.............................................................. xviii 1 General Introduction............................................................ 1 1.1 Introduction to Trypanosoma brucei..................................... 2 1.1.1 Phylogeny of Trypanosoma brucei .................................. 2 1.1.2 Trypanosomiasis and disease control ............................... 4 1.1.3 Life Cycle of Trypanosoma brucei .................................. 5 1.2 Cellular and genome organisation of Trypanosoma brucei ........... 6 1.2.1 Trypanosoma brucei cell structure ................................. 6 1.2.2 The nuclear genome structure ...................................... 7 1.3 Antigenic variation in T. brucei........................................... 9 1.4 The T. brucei cell cycle ................................................... 11 1.5 Origins of DNA replication ................................................ 13 1.5.1 Bacterial origins....................................................... 13 1.5.2 Eukaryotic origins..................................................... 15 1.5.3 Archaeal origins ....................................................... 18 1.5.4 Trypanosomatid origins .............................................. 19 1.6 The DNA replication initiation machinery; a synopsis................ 24 1.6.1 DNA replication initiation factors in bacteria.................... 25 1.6.2 DNA replication initiation factors in eukaryotes................. 27 1.6.3 Components of eukaryotic DNA replication initiation machinery ………………………………………………………………………………………………….30 1.7 DNA replication initiation in archaea ................................... 40 1.8 DNA replication initiation in T. brucei.................................. 42 1.9 Project aims................................................................. 46 1.9.1 Specific aims .......................................................... 46 2 Material and Methods ..........................................................47 2.1 Trypanosome Strains,
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