Investigating initial DNA unwinding during budding yeast replication

DNA unwinding is a central, but poorly understood process that facilitates the establishment of bi-directional replication. Sustained DNA unwinding is carried out by the replicative helicase. Most helicases require some single-stranded or untwisted DNA in order to function, and it is this initial DNA unwinding that this study aimed to characterise. There are two possible timepoints when DNA unwinding could occur: during loading of the helicase onto DNA or during the activation of that helicase, as the replisome forms. During this project each possible timepoint for initial unwinding was investigated in chronological order in vitro and S phase was scanned for the appearance of DNA unwinding in vivo. Techniques for in vitro DNaseI, nuclease P1 and potassium permanganate footprinting using supercoiled plasmid DNA were established within the first part of this project. These were implemented to reveal that no DNA unwinding occurs during helicase loading, but on completion of loading origin DNA is distorted. The latter part of this study comprised two methods for investigating DNA unwinding during helicase activation. The first method employed an in vitro approach to reconstitute the process of helicase activation during replisome formation. Here, purified proteins were used to supplement S phase yeast extracts. These mixtures were incubated with helicase pre-loaded onto origin DNA and the resulting complexes analysed using Western blotting. A novel in vivo footprinting approach, designed to access potentially unstable unwound DNA within the yeast nucleus was also executed. A single-stranded DNA-specific nuclease was expressed within yeast to target unwound DNA. DNA was extracted throughout S phase and analysed by primer extension. This study highlighted many of the obstacles associated with characterising DNA unwinding. Nevertheless, it reports the development of in vitro and in vivo assays for DNA unwinding. In addition, several new reagents were produced that will benefit future studies

Molecular characterisation of HELQ helicase’s role in DNA repair and genome stability

Maintenance of genome stability is a critical condition that ensures that daughter cells acquire an accurate copy of the genetic information from the parental cell. DNA replication stress that arises from blocked replication forks, can be a major challenge to genome integrity. Cells have therefore developed complex mechanisms to detect and deal with the replication-associated DNA damage. Intra-S-phase ATR checkpoint, FA pathway and RAD51 paralog BCDX2 complex together constitute key components of the replication stress response system that is essential to sense, repair and restart damaged forks. Previous studies in D. melanogaster and C. elegans have positioned HELQ as an important factor in DNA damage repair and maintaining genome stability. In this work I develop and combine biochemical assays, proteomic studies, mouse model and molecular biology tools to further characterise HELQ function in DNA repair and genome stability. I establish that HELQ plays a pivotal role in the replication stress response in mammalian cells. By developing a system in which I was able to pull down tagged HELQ and subject it to Mass Spectrometry analysis I identified its molecular partners and showed that HELQ interacts with, and interfaces between, the central FANCD2/FANCI heterodimer and the downstream RAD51 paralog BCDX2 complex. From mechanistical point of view, interaction with BCDX2 complex was the most interesting one and I took comprehensive experimental approach to identify the nature of HELQ-BCDX2 binding. I was able to show that this interaction does not need any mediating factors and is most likely a direct one. Despite many attempts and trying various tools I was not able to identify interacting motif neither in HELQ nor RAD51 paralogs, I identified however a candidate amino acid stretch within RAD51B as potential candidate. This works requires further validation. Additionally, I show that HELQ is actively recruited to chromatin upon exposure to DNA damaging agents and this chromatin enrichment is ATR-dependent. Once present on chromatin HELQ performs its function to promote Homologous Recombination-depended repair as shown by persistence of chromatin RAD51 and RPA32 protein in HELQ deficient cells as well as decreased HR-dependent repair measured by DR-GFP assay. To better understand how HELQ promotes HR-dependent ICL repair I decided to set up immune-depletion based ICL-repair system in Xenopus laevis egg extract (developed by Johannes Walter group). Although important progress has been made and I produced several valuable tools I did not manage to validate the system due to time limitations. Lastly, our data positions HELQ as an ovarian cancer susceptibility gene with low penetrance effect. This makes HELQ and interesting object of familial cancer screens and potentially opens an exciting therapeutic approach based on PARP inhibition

Epstein-Barr Virus episome replication and transcription

Master'sMASTER OF SCIENC

Investigating telomerase regulation in human breast cancer cells: a search for telomerase repressor sequences localised to chromosome 3P

This thesis was submitted for the award of Doctor of Philosophy and was awarded by Brunel University LondonCellular immortality is one of the ten hallmarks of human cancer and has been shown to be an essential prerequisite for malignant progression (Hanahan and Weinberg., 2011, Newbold et al., 1982, Newbold and Overell., 1983). In contrast, normal human somatic cells proliferate for a limited number of population doublings before entering permanent growth arrest known as replicative senescence. This is thought to be due to the progressive shortening of telomeric sequences with each round of cell division. Over 90% of human tumours, but not the majority of human somatic cells, have been found to express telomerase activity (Kim et al., 1994). The rate-limiting component of the human telomerase enzyme is the telomerase reverse transcriptase subunit, which is encoded by the hTERT gene. Transfection of hTERT cDNA into normal human fibroblasts and epithelial cells may sometimes be sufficient to confer cellular immortality (Newbold., 2005, Stampfer and Yaswen., 2002). Therefore, de-repression of hTERT and telomerase re-activation are thought to be critical events in human carcinogenesis and is the predominant mechanism by which cancer cells maintain their proliferative capacity. Previously, our group has shown that introduction of a normal, intact copy of human chromosome 3 into the 21NT primary breast cancer cell line by microcell-mediated monochromosome transfer (MMCT), is associated with strong telomerase repression and induction of cell growth arrest within the majority of hybrid clones (Cuthbert et al., 1999). Structural mapping of chromosome 3 within telomerase-positive revertent clones revealed two regions of deletion: 3p21.3-p22 and 3p12-p21.1, thought to harbour the putative telomerase repressor sequence(s). Subsequent studies showed that the chromosome 3p-encoded telomerase repressor sequence(s) mediates its function by means of transcriptional silencing of hTERT, in part, through chromatin remodelling of two sites within intron 2 of the hTERT gene (Ducrest et al., 2001, Szutorisz et al., 2003). Attempts to achieve positional cloning of hTERT repressor sequences on chromosome 3p identified two interesting candidates; the histone methyltransferase SETD2 and an adjacent long non-coding RNA (lncRNA) sequence known as FLJ/KIF9-AS1 (Dr. T. Roberts, unpublished data). Through MMCT-mediated introduction of intact chromosomes 3 and 17 into the 21NT cell line, I have demonstrated that at least two as yet unidentified telomerase repressor sequences (one located on each of these two normal chromosomes) may function to repress telomerase activity within the same breast cancer cell line, which suggests that multiple, independent telomerase regulatory pathways may be inactivated within the same cancer type. Furthermore, by examining the consequences of forced SETD2 and FLJ expression within the 21NT cell line, together with siRNA-mediated knockdown of SETD2 within a single telomerase-repressed 21NT-chromosome 3 hybrid, I have provided evidence to show that neither of these two candidate genes may function as a regulator of hTERT transcription. Through interrogation of relevant literature, a set of four candidate 3 telomerase regulatory genes (BAP1, RASSF1A, PBRM1 and PARP-3) were selected for further investigation based on their location within the 3p21.1-p21.3 region together with their documented role in the epigenetic regulation of target gene expression. Using mammalian expression vectors containing candidate gene cDNA sequences, my colleague Dr. T. Roberts and I demonstrated that forced overexpression of BAP1 and PARP-3 within the 21NT cell line is associated with consistent, but not always sustained, repression of hTERT transcriptional activity and telomerase activity. It is therefore possible that at least two sequences may exist on chromosome 3p that function collectively to regulate hTERT expression within breast cancer cells. Finally, using an in vitro model of human mammary epithelial cell (HMEC) immortalization, involving the targeted abrogation of two pathologically relevant genes, p16 and p53 to generate a series of variant clones at different stages of immortal transformation (developed by my colleague Dr. H. Yasaei), I have shown that single copy deletions on chromosome 3p are a frequent, clonal event, specifically associated with hTERT de-repression and immortal transformation. Subsequent high-density single nucleotide polymorphism (SNP) array analysis of immortal variants carried out by Dr. H. Yasaei, identified a minimal common region of deletion localized to 3p14.2-p22. Together, these findings provide additional evidence to show that chromosome 3p may harbour critical hTERT repressor sequences, that are lost as an early event during breast carcinogenesis

Regulation and Function of p53 in Embryonic Stem Cells

The p53 protein is one of the most well-known tumor suppressor proteins, and it plays a variety of functions in somatic cells. Once activated, p53 induces cell cycle arrest and inhibits cell proliferation. Since it was found that p53 is highly expressed in murine embryonic stem cells, a cell type that proliferates very fast because of a shortened G1 phase, it remained a mystery whether p53 is active in this cell type. I observed that a significant part of p53 is localized in the nucleus of murine embryonic stem cells and that the majority of this nuclear p53 is bound to DNA. In addition, the anti-proliferative activity of p53 is compromised in stem cells, and this control is due, at least in part, to the high amount of MDMX that is present in embryonic stem cells. This high amount of MDMX is most likely due to exclusion of exon 7 of the MDMX RNA during retinoic acid induced differentiation. MDMX co-eluted with p53 from sucrose gradient assays and downregulation of MDMX in mESCs increased MDM2 abundance, a transcriptional target of p53, indicating that MDMX controls p53’s transcriptional activity in stem cells. P53 is posttranslationally modified in mESCs and these modifications endow a neutral isoelectric point (pI) of a fraction of the p53 protein that is only present in stem cells. Moreover, according to its nuclear localization in mESCs, p53 influences the transcriptome of mESCs. However, in contrast to the anti-proliferative activity that p53 has in differentiated cells, p53 controls transcription of pro-proliferative genes in embryonic stem cells including c-myc and c-jun. Chromatin-Immunoprecipitation showed that p53 binds to the responsive element of these proto-oncogenes. The impeded anti-proliferative activity of p53 and the induction of certain proto-oncogenes by p53 in murine embryonic stem cells can explain why stem cells proliferate efficiently despite having high levels of p53

Insulin-like growth factor 2: Escape from imprinted regulation in the choroid plexus of the mouse

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

P1 nuclease defines a subpopulation of active SV40 chromatin--a new nuclease hypersensitivity assay.

Under exhaustive digestion conditions P1 nuclease was found to cleave a subpopulation of intracellular SV40 chromatin only once. The major P1 cleavage site in SV40 DNA was mapped at the origin of DNA replication, and the two minor sites at the SV40 enhancers. The P1-sensitive SV40 chromatin subpopulation was found to have higher superhelical density than the bulk of the intracellular SV40 chromatin. Furthermore, pulse labeled SV40 DNA which had higher superhelical density than that of the steady state viral DNA (S.S.Chen and M.T.Hsu, J.Virol 51:14-19, 1984) was also found to be preferentially cleaved by P1 nuclease. These results are consistent with a supercoil-dependent alteration of chromatin conformation near the regulatory region of the viral genome that can be recognized by P1 nuclease. Since P1 nuclease cleaves the subpopulation of SV40 chromatin only once without further degradation, this nuclease can be used as a general tool to define viral or cellular chromatin fraction with altered chromatin conformation and to map nuclease hypersensitive sites. Preliminary studies indicate that P1 makes limited double stranded cleavages in cellular chromatin to generate large DNA fragments

New insights into pain mechanisms through the study of genes associated with monogenic pain disorders

Pain is an intrinsic mechanism that promotes our survival by helping us to avoid injury. However, chronic pain remains a significant clinical burden and remains poorly treated. The development of new analgesic drugs may significantly improve quality of life for chronic pain patients. This thesis investigates the mechanisms of pain sensation and also suggests some novel analgesic drug targets by using molecular, genetic, and transgenic approaches. Firstly, a novel function of sodium channel Nav1.7 is explored. Microarray data showed that gene expression profiles are dramatically altered in dorsal root ganglia from Nav1.7 null mice. These changes were confirmed by real-time qRT-PCR. Altered expression of preproenkephalin (Penk) and carcinoembryonic antigen-related cell adhesion molecule 10 (Ceacam10) may contribute to the pain insensitive phenotype seen in Nav1.7 nulls. The gene expression changes were further explored using in vitro cell based assays, showing a potential role of sodium ions in controlling transcription of Penk. Secondly, we study a family with six members affected with a pain insensitive phenotype characterized by multiple painless bone fractures and frequent painless lesions caused by burning stimuli. A novel point mutation in ZFHX2, encoding a putative transcription factor expressed in small diameter sensory neurons, was identified. By analysing Zfhx2 knockout and BAC transgenic mice bearing the orthologous mutation, we confirm that ZFHX2 is crucial for normal pain perception. We study how the mutation disrupts ZFHX2 function, resulting in altered downstream expression of pain-related genes. Thirdly, a patient with small fibre neuropathy and erythromelalgia-like symptoms was genetically analysed. Using exome sequencing and detailed bioinformatics analyses, I have shortlisted three missense mutations in the genes CWC22, TMEM8B and ATL3 that are potentially pathogenic. By studying genes mutated in families with rare inherited pain disorders, this thesis broadens our understanding of pain sensation and highlights new routes to develop better analgesic drugs

The Mechanisms of DNA Replication

DNA replication is a fundamental part of the life cycle of all organisms. Not surprisingly many aspects of this process display profound conservation across organisms in all domains of life. The chapters in this volume outline and review the current state of knowledge on several key aspects of the DNA replication process. This is a critical process in both normal growth and development and in relation to a broad variety of pathological conditions including cancer. The reader will be provided with new insights into the initiation, regulation, and progression of DNA replication as well as a collection of thought provoking questions and summaries to direct future investigations