No description available.

A thesis submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfillment of the requirements for the degree of Doctor of Philosophy February 2013 / Mycobacterium tuberculosis is presented with environmental host assaults that damage its DNA during infection. Tubercle bacilli possess mechanisms to protect against moststresses imposed by the host, including genotoxic stress. However, tolerance of DNA lesions that have escaped the normal repair processes requires the function of specialist DNA polymerases that can introduce mutations during translesion synthesis (replication by-pass), thus leading to damage-induced mutagenesis. Mycobacteria employ a novel DNA polymerase, DnaE2, for DNA damage tolerance and induced mutagenesis. DnaE2 belongs to the C-family of DNA polymerases, which are known to replicate DNA with high fidelity, and has been implicated in virulence and the emergence of rifampicin resistance of M. tuberculosis in vivo. In this study, DnaE2 was shown to function in the same pathway as two accessory proteins, ImuB and ImuA’, for damage tolerance and induced mutagenesis in mycobacteria. In this system, DnaE2 performs the polymerase function in translesion synthesis whereas ImuB is a cryptic Y-family DNA polymerase that lacks critical active site residues. It contains a β-clamp binding motif that allows interaction with the β-clamp and presumably enables DnaE2 and ImuA’ to access the replication fork. ImuB has a C-terminal region extending from the β-clamp binding motif which contains disordered regions that allow the interaction with other proteins and is important for function. ImuA’ is also essential for damage tolerance and induced mutagenesis but its function remains unknown. This protein is structurally similar to Escherichia coli RecA protein in the N-terminus and the middle domain, but it has a distinct C-terminus that was shown to be important for the interaction with ImuB. The essential replicative, C-family polymerase, DnaE1, was shown to be upregulated in response to DNA damage and was also shown to interact with ImuB. To explore the possibility that other proteins are involved in this pathway, ImuB was Cterminally tagged for use as bait in pull-down experiments in M. smegmatis. However, introduction of the tag disrupted ImuB function, further reinforcing the importance of the Cterminal region of ImuB for the function of this protein, presumably via protein-protein interactions. In contrast, a variant of ImuA’ which was N-terminally tagged was shown to retain functionality; however, experiments using this protein as a bait for pull-down proved to be unsuccessful. Proteomic analysis of wild type M. smegmatis, a dnaE2 deletion mutant and complemented derivative was carried out on cells exposed to the same conditions as used in the pull-down assay. Base excision repair (BER) components were identified in this analysis, but did not detect ImuB and ImuA’, suggesting that the levels of expression of these proteins were comparatively lower under the conditions tested resulting in failure of the pull-down experiment. Finally, numerous attempts were made to express and purify recombinant forms of ImuB and ImuA’ in E. coli for use in structural studies. Both proteins were expressed in the soluble and insoluble fractions; however the levels of soluble protein were low, and as a result, purified protein preparations could not be obtained.

by Wai Ping Fung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1980. / Bibliography: leaves 214-216.

No description available.

archives@tulane.edu / The bulk of nuclear DNA synthesis during replication of the eukaryotic genome is carried out by three DNA Polymerases (Pols): Pols α, δ and ε. Through its role in leading strand synthesis, Pol ε is responsible for replicating up to half of the genome. As such, DNA synthesis errors made Pol ε during replication or other DNA repair processes pose a considerable source of potential genomic mutagenesis. Pol ε normally displays a high degree of fidelity, which can be attributed to the action of two physically distinct sites of catalysis: the polymerase domain, which is responsible for catalyzing the preferential addition of an incoming deoxynucleoside triphosphate (dNTP) to a nascent DNA strand resulting in correct Watson-Crick base pairing and the 3’ to 5’ exonuclease domain that proofreads the nascent DNA strand through the removal of misincorporated bases. Mutations in either of these domains that adversely affect their function can lead to a decrease in replication fidelity. Indeed, mutations localized to the exonuclease domain of Pol ε have been observed in tumors bearing drastically elevated genomic mutation burdens. We set out to determine the contribution of individual Pol ε mutants to mutagenesis and mutation spectrum through a combination of in vitro biochemistry and cell culture. We show that even in the face of functional mismatch repair (MMR) these mutants can lead to a variable yet substantial level of mutagenesis and recapitulate some but not all aspects of the anticipated mutation spectrum. These results indicate that Pol ε exonuclease domain mutants are capable of making these errors but other factors may be necessary to achieve the entirety of the observed patient tumor mutation profiles. In a mouse model heterozygous for the most recurrent Pol ε mutant we observe a massive reduction in tumor-free survival (100% mortality at 10 months) comprise exclusively of lymphomas. Additionally, the tumor mutation spectrum reveals a significant bias for TCT>TAT, TCG>TTG and TTT>TGT errors. These data suggest that replication errors made by Pol ε are directly contributing to tumorigenesis and may be solely responsible for the Pol ε mutant mutation profile. / 1 / Karl Hodel

Graduation date: 1997

Insects detect specific chemicals in the environment with olfactory receptors (ORs), which represent a novel class of ligand-gated ion channel. Insect ORs are comprised of at least one common subunit (OR83b in Drosophila) and at least one odorant-binding subunit. However, the molecular details of insect OR architecture, such as how they bind odorants, are unknown. This lack of knowledge hinders the development of compounds that may modulate OR function and potentially control insects involved in disease propagation and agricultural damage. The intent of this project is to investigate the structure and function of insect ORs. To this end, the utility of the Xenopus oocyte heterologous expression system was explored. Assay optimization, accuracy, and investigations on functional requirements were first performed using the Drosophila OR (DmOR) 35a/83b. The utility of the assay system was also demonstrated by identification of the honey bee (Apis mellifera) OR 11/2 as a receptor for the queen pheromone, 9-oxo-2-decenoic acid. A series of DmORs was cloned and expressed in Xenopus oocytes and individual receptors were selected for further study. DmOR85a/83b was shown to possess an incredibly high degree of enantioselectivity for the odorant ethyl 3-hydroxybutyrate. The receptive range of DmOR67a/83b was explored and observations were made on potential features of the odorant-binding site and a ligand odorophore. DmORs were also used to investigate the contributions of individual subunits toward the odorant-binding site and pore structure. Also, evidence for receptor antagonism by odorants was revealed. DmORs were screened with methanethiosulfonate reagents and the substituted cysteine accessibility method to identify residues 146-150 of DmOR85b as functionally important in receptor activation. This region, located at the predicted interface between transmembrane segment 3 (TMS3) and extracellular loop 2, was shown to be physically adjacent to the odorant-binding site itself. Finally, residues within the extracellular half of TMS3 in DmOR85b were implicated in odorant-induced activation by screening DmOR85b mutants for altered ligand preferences. Therefore, this project provides the first identification of insect OR subunit components involved in odorant recognition, and represents an important starting point for detailed analysis of the molecular basis for insect OR activation by odorants.

published_or_final_version / Zoology / Master / Master of Philosophy

No description available.

No description available.