RbpA is a RNA polymerase-binding protein that was identified in Streptomyces coelicolor. It is found in all Actinobacteria, including the pathogenic agent Mycobacterium tuberculosis. Streptomyces strains that have an rbpA mutation grow at a slower rate than the wild-type and are more sensitive to the RNAP-targeting antibiotic, rifampicin. RbpA binds to and activates σHrdB, the principal sigma factor that directs transcription of most housekeeping genes in S. coelicolor. Using bacterial two-hybrid analysis and in vitro pull down assays, RbpA was shown to interact with region 1.2-2.4 of σHrdB. This region forms part of a major interface with core RNA polymerase and is involved in the recognition of, and binding to, the -10 promoter element. Rv2050, the homologue of RbpA in M. tuberculosis, was also shown to interact with the principal sigma factor of this organism, σA. Structural studies on RbpA and Rv2050 revealed that it is composed of two regions, a structured N-terminal β-fold region and an flexible or unstable C-terminal region, which interacts with sigma. Alanine-scanning site-directed mutagenesis on the C-terminal region of RbpA identified important residues involved in σHrdB interaction as well as residues that might be involved in transcriptional activation.

Epstein Barr Virus (EBV) is a γ-herpesvirus infecting around 95% of the human population. EBV infection is life-long and asymptomatic in the majority of individuals, however EBV is associated with Nasopharyngeal Carcinoma, Burkitt's lymphoma and Hodgkin's lymphoma. The transcription factor Zta is an immediate early gene of EBV able to reactivate the virus from latency, cause cell cycle arrest and bind to sequence specific DNA elements. Cercopithecine herpesvirus-15 is a closely related virus, infecting rhesus monkeys, with a homologue to Zta; ZtaRh. A comparison of features of these proteins may be informative about critical residues in each protein. Binding to almost all response elements is conserved between the two proteins. A Zta response element (ZRE3Rh) in the CeHV-15 Rta gene promoter that is not functional for either protein was identified. ZRE3 from EBV is methylation dependent for Zta binding. Analysis of ZRE3 using competition EMSA assays has shown the importance of methylation of individual CpG motifs. ZtaRh is compromised in reactivating EBV from latency and this appears to be mediated by changes at the extreme C-terminus. ZtaRh is unable to cause G1 cell cycle arrest, however this function maps to the transactivation domain. Known binding partners of Zta were cloned to enable investigation of binding by ZtaRh or other mutants. Co-transfection of p53 and Zta resulted in rapid degradation of both proteins. Co-transfection of C/EBPα and Zta produced a larger additional Zta species. Neither effect was seen with a Zta CT mutant. RNA from HEK293-ZKO cells transfected with Zta or a C-terminal Zta mutant was analysed using a QPCR array probing 595 human genes associated with cancer, revealing possible host cell proteins influenced by Zta. Further information on the precise mechanisms of Zta could contribute to the development of future therapies for the prevention or treatment of EBV related diseases.

No description available.

Single-strand breaks (SSBs) are one of the most common types of lesion arising within cells; formed by attack of genotoxic agents on the DNA, as well as enzymatically during normal cellular processes. Although the single-strand break repair (SSBR) pathway is relatively well characterised, and many components have been extensively studied in vitro, little is known of how this pathway operates in vivo when DNA is complexed with histone proteins to form chromatin. This compaction of the DNA into nucleosomal structures has the potential to inhibit repair, by sterically blocking access of repair factors to sites of DNA damage. Whilst previous studies have shown that repair of DNA double-strand breaks and UV-induced lesions are associated with alterations in chromatin structure, through covalent modification of histone proteins and nucleosome remodeling, few similar observations have been made concerning SSBR. Here, I have produced and employed mammalian cell lines stably expressing fluorescently-tagged histone proteins to analyse the dynamics of chromatin occurring upon DNA damage. Localised damage was introduced using micro-irradiation with a UV-A laser, and the histone proteins at the site of damage visualized in real-time using confocal microscopy. Through this method, I have identified a rearrangement of chromatin structure in the vicinity of DNA strand breaks in mammalian cells, resulting in a mobilization of histone proteins at the site of damage. Furthermore, I have shown that this alteration is partially dependent on the activities of both the SSBR factor poly(ADP-ribose) polymerase 1 (PARP-1), and the phosphoinositide 3-kinase-like kinase (PIKK) Ataxia telangiectasia mutated (ATM). I have examined a potential requirement for ATM in SSBR, and found no evidence of this, suggesting that the effects of PARP-1 and ATM on histone mobilization are reflective of the independent contributions of repair of single- and double-strand breaks respectively.

No description available.

Epstein-Barr virus (EBV) is a member of the γ-herpesvirus subfamily of Herpesviridae. EBV is a double stranded DNA virus infecting humans causing a variety of disease from asymptomatic infection to association with certain tumours including Burkitts lymphoma, Hodgkin's disease and nasopharyngeal carcinoma. EBV encodes an immediate-early protein called Zta (BZLF1, EB1, ZEBRA), which is an important transcription factor and replication factor direct in disrupting latency. EBV encodes viral proteins that assemble as a replisome at the viral lytic origin recognition site (Ori-Lyt). Zta binds Ori-Lyt and it is unclear how Zta interacts and recruits the complex to the site of DNA replication, while coordinating and recruiting host factors. After a mutation to three alanines (ZtaAAA) data implicates that the extreme C-terminus of Zta is essential for replication. The question posed is how does Zta assemble the replisome? Identification of the lytic changes that contribute to lytic replication, including cellular components that may contribute to EBV replication is attempted. Transfected control, Zta and ZtaAAA in HEK293-BZLF1-KO cells was compared. Size exclusion chromatography identified a higher molecular weight complex containing Zta during viral replication. SILAC (Stable isotope labelling by amino acids in cell culture) coupled to proteomics analysis identified the elution fraction composition. An interpretation of these cellular components in the context of lytic replication is explored. Identification of interactions of Zta with cellular proteins was attempted by SILAC histidine tagged Zta with pull down assay. Quantitative data was returned and a confirmation of interactions was attempted. A global proteomics approach was also performed. An enrichment method to isolate SILAC labeled Burkitts Lymphoma cells undergoing EBV lytic replication was coupled to mass spectrometry analysis to identify changes in host and viral proteins. Overall, cellular targets that may interact with Zta are to be confirmed. The global proteomics study recognized for the first time by proteomic analysis the identification of three EBV lytic replication cycle proteins.

Recent studies into the stringent response and the discovery of a number of RNA polymerase binding proteins suggests that the model for bacterial transcription initiation in Actinobacteria may differ from that in Escherichia coli. In E. coli, the alarmone ppGpp, together with DksA, binds to RNA polymerase to elicit the stringent response. However, the ppGpp binding site on RNA polymerase is not conserved in S. coelicolor, although the organism possesses a DksA homologue. Deletion of DksA did not affect the growth and development of S. coelicolor, although its overexpression stimulated antibiotic production. Evidence is presented that suggests that this occurs through binding to the RNA polymerase secondary channel. The biological role of this protein remains unknown. CarD and RbpA are two RNA polymerase­‐binding proteins present in all Actinobacteria, including S. coelicolor and M. tuberculosis. Both proteins are critical for growth and have been identified as transcriptional activators from σHrdB­‐dependent promoters in vitro. Here it was demonstrated that CarD and RbpA activate transcription from rRNA promoters with a poorly conserved ­‐35 element. Surprisingly it was also found that both proteins can inhibit transcription from synthetic promoters with highly conserved ­‐35 elements. Chromatin immunoprecipitation followed by high throughput sequencing (ChIP­‐seq) experiments revealed that CarD and RbpA are found exclusively at promoter regions. RbpA is localised only at promoters recognised by σHrdB, whereas CarD also co‐localises with the alternative sigma factor σR during oxidative stress indicating that it lacks RNA polymerase holoenzyme specificity. The sigma specificity of RbpA was tested by the generation of sigma mutants that were defective in binding. In vivo, in vitro and ChIP­‐seq data presented in this study suggest that CarD and RbpA have an overlapping role in transcription initiation at σHrdB­‐dependent promoters in S. coelicolor.

Nucleotide excision repair is one of the major repair pathways responsible for identifying and removing lesions in the DNA double helix. In higher eukaryotes, nucleotide excision repair is a coordinated response of over 30 proteins recruited in an ordered procession with distinct roles in the recognition, removal and repair of said lesions. A key step in the completion of the repair process is the resynthesis of the excised strand using the undamaged partner as a template. DNA polymerase kappa (polκ), a member of the Y-family, has been shown to have a role in nucleotide excision repair distinct from its traditional role in translesion synthesis. Cell lines lacking polκ showed clear defects in nucleotide excision repair and increased ultraviolet light sensitivity. Building on this established work, conserved residues were identified in the C-terminus of human polκ and mutated to alanines. Under transient expression, mutations in the ubiquitin binding domains severely impaired the recruitment to sites of damage. Cell lines defective in polκ that stably expressed these mutant polymerases showed sensitivity to ultraviolet radiation following exposure; intriguingly, this defect seems confined to the global genomic repair pathway as no substantial defect in transcription-coupled repair was observed. Following on from these observations, immunoprecipitation of the polymerase and partner proteins was investigated in an attempt to identify interactions disrupted by the mutations to the ubiquitin binding domains. These experiments indicated impairment in binding to ubiquitinated PCNA in the mutants. In further work, the recruitment of wild-type human polκ was shown to be independent of the 3' incision by the nuclease XPG during the repair process, consistent with a recently proposed model for NER.

DNA replication is a central mechanism to all forms of life. Errors occurring during DNA replication can result in mutagenesis and genome rearrangements, which can cause various diseases. In this work I have investigated the stability of direct tandem repeats (TRs) in the context of replication and replication-associated repair mechanisms. During DNA replication the replication fork encounters many obstacles, such as DNA-protein barriers, secondary DNA structures and DNA lesions. How and if replication resumes or restarts in these circumstances in order to complete genome replication is not well understood and the fidelity of replication in response to such obstacles remains unclear. I have developed TR assays to assess replication errors in the context of replication fork restart and secondary structures. The results suggest that structured DNA (G4) can cause instability of TRs in the context of normal replication and that restarted replication can be intrinsically error-prone. Surprisingly, the mutagenic effect of G4-DNA on TR stability was not elevated in the context of replication fork restart. Therefore, deletions of TRs containing G4-DNA are not more susceptible to the compromised fidelity of a restarted replication fork. Structures such as stalled replication forks can induce checkpoint responses to maintain genome stability. The stabilisation of replication forks is central in the response to replication stress. These protective mechanisms include the regulation of enzymatic activities. Mus81-Eme1 is a structure-specific endonuclease which is regulated by the DNA replication checkpoint, but has also been shown to be required for replication fork restart in certain circumstances. In collaboration with Professor Neil McDonald I analysed a novel domain identified in Mus81-Eme1. Mutagenesis of key residues deduced from the protein structure and comparison of their genetic analysis to known phenotypes of Mus81-Eme1 suggests distinct requirements for this domain.

The aim of this project was to investigate the effects of a novel mouse cytoplasmic dynein mutation; Abnormal rear leg (Arl). Cytoplasmic dynein is a microtubule (MT) based motor protein important for diverse cellular processes including Golgi maintenance and retrograde transport of organelles. Arl is a mouse point mutation in the heavy chain subunit of dynein (Dync1h1). Homozygous Dync1h1Arl/Arl die at embryonic day 10. Dync1h1Arl/+ heterozygotes have a normal life span, but exhibit abnormal gait and hindlimb clasping during tail suspension, typical of neuronal dysfunction. Protein purification from wildtype and heterozygous brain tissue showed increased MT binding in Dync1h1Arl/+ compared to wildtype. Delayed endosomal trafficking was observed in EGF stimulated Dync1h1Arl/+ mouse embryonic fibroblasts (MEFs) compared to wildtype, in both fixed cells and using live cell imaging. Similarly, a delay in the reassembly of the Golgi complex after disruption with a MT depolymerisation agent, nocodazole, was observed in Dync1h1Arl/+ MEFs compared to wildtype. In addition, the Golgi complex was observed as being structurally perturbed in Dync1h1Arl/+ lumbar spinal cord neurons using transmission electron microscopy (TEM) compared to the wildtype. TEM also revealed that the mitochondria were structurally perturbed in Dync1h1Arl/+ lumbar spinal cord neurons compared to wildtype, and O2 consumption assays investigating their function showed the Dync1h1Arl/+ mitochondria to have increased respiration rates compared to wildtype. Thus, these data highlight the Arl mouse as an invaluable model for studying the mechanism of dynein function and the subsequent outcomes when they are compromised.