Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 71-80).

Ribonucleotide reductase (RNR) is a key enzyme in the de novo biosynthesis and homeostatic maintenance of all four DNA building blocks by being able to make deoxyribonucleotides from the corresponding ribonucleotides. It is important for the cell to control the production of a balanced supply of the dNTPs to minimize misincorporations in DNA. Because RNR is the rate-limiting enzyme in DNA synthesis, it is an important target for antimicrobial and antiproliferative molecules. The enzyme RNR has one of the most sophisticated allosteric regulations known in Nature with four allosteric effectors (ATP, dATP, dGTP, and dTTP) and two allosteric sites. One of the sites (s-site) controls the substrate specificity of the enzyme, whereas the other one (a-site) regulates the overall activity.  The a-site binds either dATP, which inhibits the enzyme or ATP that activates the enzyme. In eukaryotes, ATP activation is directly through the a-site and in E. coli it is a cross-talk effect between the a and s-sites. It is important to study and get more knowledge about the overall activity regulation of RNR, both because it has an important physiological function, but also because it may provide important clues to the design of antibacterial and antiproliferative drugs, which can target RNR. Previous studies of class I RNRs, the class found in nearly all eukaryotes and many prokaryotes have revealed that the overall activity regulation is dependent on the formation of oligomeric complexes. The class I RNR consists of two subunits, a large α subunit, and a small β subunit. The oligomeric complexes vary between different species with the mammalian and yeast enzymes cycle between structurally different active and inactive α6β2 complexes, and the E. coli enzyme cycles between active α2β2 and inactive α4β4 complexes. Because RNR equilibrates between many different oligomeric forms that are not resolved by most conventional methods, we have used a technique termed gas-phase electrophoretic macromolecule analysis (GEMMA). In the present studies, our focus is on characterizing both prokaryotic and mammalian class I RNRs. In one of our projects, we have studied the class I RNR from Pseudomonas aeruginosa and found that it represents a novel mechanism of overall activity allosteric regulation, which is different from the two known overall activity allosteric regulation found in E. coli and eukaryotic RNRs, respectively.  The structural differences between the bacterial and the eukaryote class I RNRs are interesting from a drug developmental viewpoint because they open up the possibility of finding inhibitors that selectively target the pathogens. The biochemical data that we have published in the above project was later supported by crystal structure and solution X-ray scattering data that we published together with Derek T. Logan`s research group. We have also studied the effect of a novel antiproliferative molecule, NSC73735, on the oligomerization of the human RNR large subunit. This collaborative research results showed that the molecule NSC73735 is the first reported non-nucleoside molecule which alters the oligomerization to inhibit human RNR and the molecule disrupts the cell cycle distribution in human leukemia cells.

<p>Homing endonuclease genes (HEGs) are selfish genetic elements that mediate their own super-Mendelian inheritance. This is mediated by the homing endonuclease cleavage of a HEG^-allele followed by recombination-repair with a HEG⁺ allele.</p><p>The majority of the HEGs are encoded in intervening sequences (IVSs). The insertion of the IVS interrupts the endonuclease recognition site, making the genome with the IVS resistant to further cleavage by homing endonucleases with specificity for that particular sequence, but susceptible for homing endonucleases with a target not affected by the IVS insert. In 39 studied strains of the <i>Bacillus cereus</i> group, 28 IVSs were found in the <i>nrdIEF</i> operon. Phylogenetic studies of these sequences showed a scattered distribution of the IVSs, indicating a frequent horizontal gene transfer and that there might be competition between the different IVSs in the <i>nrdIEF</i> operon in the <i>Bacillaceae</i> family. One novel group I intron was shown to encode a functional homing endonuclease with a GIY-(X)₈-YIG motif, expanding the family motif to GIY-(X)₈-₁₁-YIG. Interestingly, by studying the known insertion sites for IVSs in the ribonuclotide reductase genes, we show that the majority of the insertions are at conserved motifs, indicating that conservation is important for IVS survival.</p><p>Most freestanding HEGs in bacteriophage T4 cleave both HEG⁺ and HEG^- alleles, possibly providing a competitive advantage for the host organism when two phages infect the same bacterium. Two novel freestanding HEGs replace two putative HEGs in T4 in some T-even-like phages. The characterisation of these HEGs showed that both cleave double stranded DNA. SegH was shown to promote homing of its gene. Hef showed no homing, possibly due to general exclusion of other phages. The <i>mobE</i> putative HEG was shown to be homing proficient and showed strong general DNA degradation when expressed in <i>Escherichia coli.</i></p>

Homing endonuclease genes (HEGs) are selfish genetic elements that mediate their own super-Mendelian inheritance. This is mediated by the homing endonuclease cleavage of a HEG- allele followed by recombination-repair with a HEG+ allele. The majority of the HEGs are encoded in intervening sequences (IVSs). The insertion of the IVS interrupts the endonuclease recognition site, making the genome with the IVS resistant to further cleavage by homing endonucleases with specificity for that particular sequence, but susceptible for homing endonucleases with a target not affected by the IVS insert. In 39 studied strains of the Bacillus cereus group, 28 IVSs were found in the nrdIEF operon. Phylogenetic studies of these sequences showed a scattered distribution of the IVSs, indicating a frequent horizontal gene transfer and that there might be competition between the different IVSs in the nrdIEF operon in the Bacillaceae family. One novel group I intron was shown to encode a functional homing endonuclease with a GIY-(X)8-YIG motif, expanding the family motif to GIY-(X)8-11-YIG. Interestingly, by studying the known insertion sites for IVSs in the ribonuclotide reductase genes, we show that the majority of the insertions are at conserved motifs, indicating that conservation is important for IVS survival. Most freestanding HEGs in bacteriophage T4 cleave both HEG+ and HEG- alleles, possibly providing a competitive advantage for the host organism when two phages infect the same bacterium. Two novel freestanding HEGs replace two putative HEGs in T4 in some T-even-like phages. The characterisation of these HEGs showed that both cleave double stranded DNA. SegH was shown to promote homing of its gene. Hef showed no homing, possibly due to general exclusion of other phages. The mobE putative HEG was shown to be homing proficient and showed strong general DNA degradation when expressed in Escherichia coli.

From microscopic bacteria to the giant whale, every single living organism on Earth uses the same language of life: DNA. Deoxyribonucleoside triphosphates––dNTPs (dATP, dTTP, dGTP, and dCTP)––are the building blocks of DNA and are therefore the “alphabet of life”. A balanced supply of dNTPs is essential for integral DNA transactions such as faithful genome duplication and repair. The enzyme ribonucleotide reductase (RNR) not only synthesizes all four dNTPs but also primarily maintains the crucial individual concentration of each dNTP in a cell. In this thesis we investigated what happens if the crucial balanced supply of dNTPs is disrupted, addressing whether a cell has a mechanism to detect imbalanced dNTP pools and whether all pool imbalances are equally mutagenic. To address these questions, we introduced single amino acid substitutions into loop 2 of the allosteric specificity site of Saccharomyces cerevisiae RNR and obtained a collection of yeast strains with different but defined dNTP pool imbalances. These results directly confirmed that the loop 2 is the structural link between the substrate specificity and effector binding sites of RNR. We were surprised to observe that mutagenesis was enhanced even in a strain with mildly imbalanced dNTP pools, despite the availability of the two major replication error correction mechanisms: proofreading and mismatch repair. However, the mutagenic potential of different dNTP pool imbalances did not directly correlate with their severity, and the locations of the mutations in a strain with elevated dTTP and dCTP were completely different from those in a strain with elevated dATP and dGTP. We then investigated, whether dNTP pool imbalances interfere with cell cycle progression and if they are detected by the S-phase checkpoint, a genome surveillance mechanism activated in response to DNA damage or replication blocks. The S-phase checkpoint was activated by the depletion of one or more dNTPs. In contrast, when none of the dNTP pools was limiting for DNA replication, even extreme and mutagenic dNTP pool imbalances did not activate the S-phase checkpoint and did not interfere with the cell cycle progression. We also observed an interesting mutational strand bias in one of the mutant rnr1 strains suggesting that the S-phase checkpoint may selectively prevent formation of replication errors during leading strand replication. We further used these strains to study the mechanisms by which dNTP pool imbalances induce genome instability. In addition, we discovered that a high dNTP concentration allows replicative DNA polymerases to bypass certain DNA lesions, which are difficult to bypass at normal dNTP concentrations. Our results broaden the role of dNTPs beyond ‘dNTPs as the building blocks’ and suggest that dNTPs are not only the building blocks of DNA but also that their concentrations in a cell have regulatory implications for maintaining genomic integrity. This is important as all cancers arise as a result of some kind of genomic abnormality.

1). The effect of temperature, P450 inhibitors (pyrazole and imidazole), sulphydryl reagents (iodoacetamide and N-ethyl maleimide) and glutathione on the activation of CCl4 in rat liver microsomes was studied. Spin trapping of CCI3', covalent binding of CCl4 to protein and CCl4-dependent MDA formation were used as indices of CCl4 metabolism. Formation of PBN-CCI3' adduct, 14CCl4 covalent binding to protein and CCl4-dependent :MDA production were dependent on temperature range from 15-40°C. The transition temperature was at 26.7 -27 .5°C when the activation was measured by formation of PBNCCl3' adduct and specific 14CCl4 covalent binding. The transition temperature was found to be 34.3°C when CCl4 -dependent MDA production was taken as the index of the activation of CCI4. Pyrazole, imidazole and iodoacetamide inhibited CC14 -dependent MDA formation only at high concentrations (10-20 mM), whereas glutathione showed a strong inhibitory effect on CCl4-stimulated lipid peroxidation. MDA formation was nearly 100°;6 inhibited by 1 roM GSH. GSH also delayed the onset of lipid peroxidation. N-ethyl maleimide (NEM) exerted biphasic effects on CCl4 -dependent MDA formation. The lower concentration of NEM (0.5 mM-l mM) reduced the :MDA prodUction, while the higher concentration of NEM (5-10 mM) enhanced the MDA formation. 2). Ribonucleotide reductase was partially purified from juvenile normal rat liver. The enzyme was purified 30 fold after DEAE-cellulose chromatography. The CDP reductase activity in tissues with different growth states or rates was compared. The enzyme activity was developed well in juvenile rat liver, regenerating liver and hepatoma (cells), while the enzyme activity was undetectable in adult rat liver and sham-operated rat liver. The enzyme activity in Yoshida cells was 3-fold of the activity in Morris 5123tc tumours. Dithiothreitol (DIT) activated the activity of CDP reductase from 48h and 60h regenerating liver, but DIT did not activate the enzyme activity of juvenile 'normal rat liver. The possible mechanism of the activation of enzyme activity by DIT was discussed and a mechanism of regulation of the ribonucleotide reductase activity in regenerating liver was suggested. 3). The effect of the lipid peroxidation product 4-hydroxynonenal (HNE) on CDP reductase from juvenile normal rat liver was investigated. HNE inhibited the CDP reductase activity. The inhibition was dependent on the concentration of HNE and the incubation time. The enzyme activity was reduced 500/0 by 0.1 roM HNE. The inhibitory effect of HNE was irreversible. DIT protected the enzyme against HNE suggesting that HNE inhibited the activity of ribonucleotide reductase from rat liver through the mechanism of blockage of functional SH groups in the enzyme protein.

Diss. (sammanfattning) Stockholm : Univ., 2007. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 132-147).

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 56-75).

Submitted by Erika Demachki (erikademachki@gmail.com) on 2014-10-31T16:54:52Z No. of bitstreams: 2 Dissertação - Elismar de Jesus Nunes Cnossen - 2014.pdf: 4126749 bytes, checksum: eacd4dd92411aa26514ec275da57a07e (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Jaqueline Silva (jtas29@gmail.com) on 2014-10-31T16:58:44Z (GMT) No. of bitstreams: 2 Dissertação - Elismar de Jesus Nunes Cnossen - 2014.pdf: 4126749 bytes, checksum: eacd4dd92411aa26514ec275da57a07e (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2014-10-31T16:58:44Z (GMT). No. of bitstreams: 2 Dissertação - Elismar de Jesus Nunes Cnossen - 2014.pdf: 4126749 bytes, checksum: eacd4dd92411aa26514ec275da57a07e (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2014-01-21 / Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG / The increasing number of notifications of dengue infections is becoming a very important concern for global healthcare programs. Combinatorial technologies aiming the selection of specific short conformational nucleic acid ligands against viral targets, also called aptamers, can be achieved by large-scale selections using the genomic SELEX technology. Our hypothesis is that aptamers can be directly selected against dengue RNA conformational structures that present functional elements in the 5'-UTR sequence, which form RNA-RNA and protein- RNA interactions, and play significant roles in the infection process. Our aim was to select and characterize aptamers that bind to the 5’-UTR using the matrix-free SELEX method. Products from the eighth selection cycle were isolated, cloned and sequenced, and 14 ligands were chosen for in silico characterization. Aptamers were grouped into three families according to their sequence homology, and conserved ribonucleotides generated specific linear motifs. Sequences motifs were detected in random nucleotides regions ranging from 31 to 40 nt, which showed higher affinity to DENV1 and 3 virus. The novel molecules and processes described in this study open new insights for dengue research and applications, and the selected aptamers can be used either as diagnostic or therapeutic tools. In silico analyses revealed that aptamer binding to its RNA target may lead to alterations of viral RNA secondary structures, and is probably leading to the loss of its original conformational and preventing its replication and/or the transcription process. The analyses also demonstrated that aptamers presented a broad hybridization spectrum to DENV1 and 3 even in the presence of mutations in different subtypes, which suggest its possible use in the other two serotypes, DENV2 and 4. This is the first description of aptamers against the RNA structure of Dengue Virus with important implications in the disease control. / O aumento do número de notificações de infecções causadas pela dengue está se tornando uma grande preocupação para os programas globais de saúde. Tecnologias combinatórias destinadas à seleção de ligantes específicos de ácidos nucléicos curtos conformacionais contra alvos virais, também chamados aptâmeros, podem ser conseguidos pelas seleções em larga escala, utilizando a tecnologia do SELEX genômico. Nossa hipótese é que os aptâmeros podem ser selecionados diretamente contra as estruturas conformacionais de RNA da dengue, as quais apresentam elementos funcionais na sequência 5'-UTR, que formam interações RNA-RNA e RNA-proteína, e desempenham papéis importantes no processo de infecção. O nosso objetivo foi selecionar e caracterizar aptâmeros que se ligam a região 5'-UTR utilizando o método matrix-free SELEX . Produtos do oitavo ciclo de seleção foram isolados, clonados e sequenciados, e 14 ligantes foram escolhidos para a caracterização in silico. Os aptâmeros foram agrupados em três famílias de acordo com a homologia das sequências, e ribonucleotídeos conservados geraram motivos lineares específicos. Sequências motivos foram detectadas em regiões aleatórias de nucleotídeos variando de 31-40 nt que apresentaram a maior afinidade para DENV1 e 3. As novas moléculas e processos descritos neste estudo abrem novas perspectivas para a pesquisa e aplicações na dengue, e os aptâmeros selecionados podem ser usados tanto como ferramentas diagnósticas ou terapêuticas. As análises in silico revelaram que a ligação do aptâmeros ao seu alvo de RNA pode levar a alterações na estrutura secundária do RNA viral, e provavelmente levando à perda da sua conformação original e impedindo a sua replicação e/ou o processo de transcrição. As análises também demonstraram que os aptâmeros apresentaram um largo espectro de hibridação ao DENV1 e 3, mesmo na presença de mutações em diferentes subtipos, o que sugere a sua possível utilização nos outros dois sorotipos, DENV2 e 4. Esta é a primeira descrição de aptâmeros contra a estrutura de RNA do Vírus da Dengue com implicações importantes no controle da doença.