Thesis (M.Sc.) -- University of Adelaide, Dept of Biochemistry, 1972.

Thesis (Ph.D.) -- University of Adelaide, Dept. of Biochemistry, 1972.

Thesis (Ph. D.)--University of Iowa, 2008. / Thesis supervisor: M. Todd Washington. Includes bibliographical references (leaves 150-163).

Thesis (Ph. D.)--University of Wisconsin--Madison, 1982. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

A small lipid-containing bacteriophage PRD1 specifies its own DNA polymerase which utilizes terminal protein as a primer for DNA synthesis. The PRD1 DNA polymerase gene has been sequenced and its amino acid sequence deduced. This protein-primed DNA polymerase consists of 553 amino acid residues with a calculated molecular weight of 63,300. Thus, it is the smallest DNA polymerase ever isolated from prokaryotic cells. Comparison of the PRD1 DNA polymerase with other DNA polymerases whose sequences have been published, yielded segmental but significant homologies. These results strongly suggest that many prokaryotic and eukaryotic DNA polymerase genes regardless of size have evolved from a common ancestral gene. The results further indicate that those DNA polymerases which use either an RNA or protein primer are related. We propose to classify DNA polymerases on the basis of their evolutionary relatedness. In order to overexpress PRD1 DNA polymerase in E. coli cells, the 2kb Hae II fragment was isolated from phage genomic DNA. This fragment was then cloned into pEMBLex3 expression vector. Phagemid pEMBLex3 contains lambda pR promoter and cI857 gene as a repressor. A specific 57 bp deletion was performed by using uracil containing ss DNA and oligonucleotide spanning each region to remove an unwanted non-coding region. After this deletion, the PRD1 DNA polymerase gene is totally under the control of the vector promoter and SD sequence. Upon heat induction, a protein with an apparent size of 68 kdal was overexpressed as an active PRD1 DNA polymerase. The expression of DNA polymerase was about 1% of total E. coli protein. The PRD1 DNA polymerase is a small multifunctional DNA polymerase and has three major conserved amino acid sequences which are shared among many DNA polymerases including human DNA polymerase alpha. Therefore, the PRD1 DNA polymerase provides an useful model system to study structure-function analysis of DNA polymerases. Four specific amino acid changes generated in conserved regions by the site-directed mutagenesis, in order to investigate their functional roles. Based on complementation test, three conserved regions are functional domains of PRD1 DNA polymerase.

Immunochemical and immunohistochemical techniques were used to determine the role of post-translational modifications in the regulation of DNA polymerase α in Rat-1(tsLA24/RSV) cells. Immunoaffinity purification following sucrose gradient fractionation showed two immunospecific polypeptides of Mᵣ ≃ 185,000 and 220,000 only in those fractions exhibiting DNA polymerase α activity. The Mᵣ ≃ 220,000 polypeptide was shown to be phosphorylated, primarily at serine residues. Incubation of cell lysates with immobilized alkaline phosphatase reduced enzyme activity and subsequent readdition of ATP, but not ATP-γ-S, restored activity suggesting the involvement of an endogenous serine protein kinase. This kinase may be a cAMP dependent protein kinase because prior incubation of the catalytic subunit stimulated DNA polymerase α activity 3-4 fold. In the absence of serum growth factors or pp60ˢʳᶜ, DNA polymerase α activity and semi-conservative DNA replication rates in growth arrested cells were severely depressed. However, both polymerase activity and DNA synthetic rates were subsequently restored by either activation of pp60ˢʳᶜ by temperature shift or by serum addition. DNA polymerase α protein was found primarily in the nucleus of all cells in log phase, growth arrested or subsequently stimulated cultures, independent of whether the cells were replicating DNA. Stimulation by either pp60ˢʳᶜ or serum did not alter DNA polymerase α localization within the cell nor lead to a preferential synthesis of Mᵣ ≃ 220,000 peptide or proteolytic conversion of the Mᵣ ≃ 220,000 peptide to smaller peptides, but did result in phosphorylation of the Mᵣ ≃ 220,000 polypeptide. This phosphorylation was not apparent in serum deprived, growth arrested cells. It is suggested that pp60ˢʳᶜ acts to initiate DNA synthesis through the temporal activation of DNA polymerase α through a mechanism similar to that used by serum growth factors and that phosphorylation by a serine protein kinase serves an important function.

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.

Treatments for the hepatitis C virus (HCV) are currently only partially effective. Research into antivirals directed at HCV viral proteins are commonly based and tested on a single genotype, namely genotype 1. This is despite the high level of variability of the RNA virus and the frequency of infection with genotypes other than 1. The systematic evolution of ligands by exponential enrichment (SELEX) is a novel in vitro approach for the isolation of antiviral agents. SELEX allows rapid screening of vast nucleic acid libraries to isolate sequences (termed aptamers) that bind to target proteins with high affinity. The SELEX approach was used in the present study to isolate DNA aptamers to the RNAdependent RNA polymerase (RdRp) [non-structural protein B (NS5B)] protein of HCV subtype 3a, with the aim of inhibiting polymerase activity. Ten rounds of selection were performed using a Biacore 2000 and resultant aptamers cloned from rounds 2, 4, 8 and 10. Sequences of aptamers were aligned to elucidate common motifs and a proportion of the aptamers from rounds 8 and 10 (29/48) were screened for binding ability using the Biacore. The five ???best binding??? aptamers were investigated for inhibition of 3a polymerase activity in an in vitro polymerase assay. Two aptamers, r10/43 and r10/47, were chosen for further studies based on their ability to inhibit polymerase activity. The inhibition constants (Ki) of r10/43 and r10/47 were estimated to be 1.4 + 2.4 nM and 6.0 + 2.3 nM respectively. The affinity (Kd) of these aptamers for the 3a polymerase was estimated to be 1.3 + 0.3 nM (r10/43) and 23.5 + 6.7 nM (r10/47). The estimated inhibition and dissociation constants of these two aptamers are among the best for inhibitory aptamers of the HCV enzymes (polymerase and protease). Inhibition of HCV 3a polymerase appeared to be specific for r10/47, whilst r10/43 also had some inhibitory effect on norovirus and ??6 polymerase activity. This study is the first description of an inhibitor to the HCV subtype 3a polymerase that investigates genotypic specificity of targeted antivirals.

Thesis (M. Med. Sc.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 99-101).