Spelling suggestions: "subject:"dna polymerase""
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Substrate recognition by the yeast Rev1 protein and dna polymerase [zeta]Howell, Craig A. January 2008 (has links)
Thesis (Ph. D.)--University of Iowa, 2008. / Thesis supervisor: M. Todd Washington. Includes bibliographical references (leaves 150-163).
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Structure-function analysis of PRD1 DNA polymerase; nucleotide sequence, overexpression and in vitro mutagenesis of the PRD1 DNA polymerase gene.Jung, Guhung. January 1989 (has links)
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.
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PHOSPHORYLATION OF DNA POLYMERASE ALPHA IN NORMAL AND ROUS SARCOMA VIRUS TRANSFORMED RAT FIBROBLASTS.DONALDSON, ROBERT WILLIAM. January 1987 (has links)
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.
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Mechanisms of mutagenesis in Mycobacterium tuberculosis: structural and functional characterisation of the DNA polymerase accessory factors encoded by Rv3394c and Rv3395cNdwandwe, Duduzile Edith 29 July 2013 (has links)
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.
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Kinetics of DNA polymerase conformational changes during nucleotide binding and incorporationTsai, Yu-chih 28 August 2008 (has links)
Not available / text
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Fidelity of nucleotide incorporation by the human mitochondrial DNA polymeraseLee, Harold Ray 28 August 2008 (has links)
Not available / text
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DNA curvature and fluctuational base pair opening in the promoter regions of escherichia coliPlaskon, Randolph Richard 12 1900 (has links)
No description available.
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Design, synthesis and evaluation of new intercalator analogs targeting human telomeraseRangarajan, Subhashree, Friedman, Simon H. January 2006 (has links)
Thesis (Ph. D.)--School of Pharmacy and Dept. of Chemistry. University of Missouri--Kansas City, 2006. / "A dissertation in pharmaceutical science and chemistry." Advisor: Simon H. Friedman. Typescript. Vita. Description based on contents viewed Nov. 9, 2007; title from "catalog record" of the print edition. Includes bibliographical references (leaves 292-317). Online version of the print edition.
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Biochemical and structural analysis of the p58C and p68N domains of DNA polymerase alpha/primaseWeiner, Brian Edward. January 2008 (has links)
Thesis (Ph. D. in Biochemistry)--Vanderbilt University, Aug. 2008. / Title from title screen. Includes bibliographical references.
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Insight into the fidelity of two X-family polymerases DNA polymerase mu and DNA polymerase beta /Roettger, Michelle P. January 2008 (has links)
Thesis (Ph. D.)--Ohio State University, 2008.
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