The first committed step in DNA biosynthesis occurs by direct reduction of ribonucleotides. This reduction is catalyzed by ribonucleotide reductase (RNR), an enzyme which uses a unique radical mechanism to facilitate the transformation. All four DNA precursors are synthesized by a single enzyme. Therefore, an intricate pattern of regulation has evolved to insure that RNR generates the proper quantity of each deoxyribonucleotide. It is this regulation, and conditions that influence this regulation, that are the central focal points of this dissertation. The studies described in this thesis have been aided by the development of a novel RNR assay. Unlike the traditional assay, this new procedure permits the simultaneous monitoring of all four RNR activities. This four-substrate assay was used to investigate whether the four enzyme activities of RNR were differentially sensitive to inhibition by the radical scavenger, hydroxyurea. The assay results, along with the results of a technique that measured enzyme inhibition as a function of radical decay, suggest that all activities of RNR are equally inhibited by hydroxyurea. Instead of differential inhibition, it appears that the activity level of RNR determines the relative sensitivity to hydroxyurea. The effects of nucleotide effectors and substrates on the relative turnover rates of the vaccinia virus and T4 phage RNR were also investigated by use of the four-substrate assay. When physiological concentrations of the allosteric effectors and substrates were added to the reaction mixtures, both enzyme forms produced dNDPs in ratios that approximate the nucleotide composition of their respective genomes. Non-physiological nucleotide concentrations generated significantly different product profiles, indicating that RNR has evolved to function within a defined nucleotide environment. Interestingly, the substrate component of the nucleotide environment proved to be as important as the allosteric effectors in modulating the reaction rates. Although the allosteric effects of nucleoside triphosphates have been known for some time, little attention has been given to the potential role that substrates play in the regulation of RNR. The results from my research suggest that the regulation of RNR in vivo results from a complex interplay between the enzyme and its substrates, products, and allosteric effectors. / Graduation date: 1998

No description available.

No description available.

Typescript (photocopy). / Digitized by Kansas Correctional Industries

Oxidative stress (OS) is strongly implicated in age-related neurodegeneration and other diseases. Under OS, the production of excessive oxidants leads to increased damages to cellular components. Recently, RNA has been discovered as a major target of oxidative damage, including the creation of abasic sites. In this work, we developed a method for quantifying abasic RNA in cell. Using this method, we have examined the potential role of the RNA-processing cellular foci, stress granule (SG) and processing bodies (PB) in eliminating abasic RNA in situ. We demonstrated that RNA is a major target of oxidative damage, constituting the majority of OS-induced abasic nucleic acids in HeLa cell. Importantly, the level of abasic RNA is strongly correlated with SG abundance. Furthermore, inhibition of SG/PB formation causes accumulation of abasic RNA, suggesting that SG/PB participates in removing oxidized RNA and protects cells under OS, which offers novel targets for therapeutic intervention in age-related diseases. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2016. / FAU Electronic Theses and Dissertations Collection

No description available.

We have studies oxidative damage of RNA, a major type of cellular macromolecules. RNA is a primary target of reactive oxygen species (ROS). Under oxidative stress, most nucleic acid damages in Escherichia coli (E.coli) are present in RNA as shown by high levels of 8-oxo-G, an oxidized form of guanine. Increased RNA oxidation is closely correlated to cell death under oxidative stress. Surprisingly, neither RNA structure nor association with proteins protects RNA from oxidation... Our results demonstrate a major role for RNA degradation in controlling oxidized RNA. We have identified activities that may work in specific pathways for selectively degrading damaged RNA. These activities may play pivotal rold in controlling oxidized RNA and protecting cells under oxidative stress. / by Min Liu. / Thesis (Ph.D.)--Florida Atlantic University, 2012. / Includes bibliography. / Mode of access: World Wide Web. / System requirements: Adobe Reader.

Doctorat en Sciences / info:eu-repo/semantics/nonPublished

RNA damage occurring under oxidative stress has been shown to cause RNA dysfunction and must be detrimental to cells and organisms. We propose that damaged RNA can be removed by specific RNA surveillance activities. In this work, we investigated the role of polynucleotide phosphorylase (PNPase), a 3'->5' exoribonuclease, in protecting the cells against oxidative stress and eliminating oxidatively-damaged RNA. Previously, it was reported that E. coli PNPase has a higher affinity to poly(8-oxoG:A). We further confirmed that E. coli PNPase can specifically bind to an oxidized RNA with a high affinity. An E. coli strain deficient in PNPase (pnp) is hypersensitive to hydrogen peroxide (H2O2). Importantly, the level of H2O2-induced RNA damage, measured by the content of 8-hydroxyguanosine, increases significantly in the pnp mutant cells. Consistent with the notion that PNPase plays a direct role in these processes, introduction of the pnp gene encoding E. coli PNPase can restore the viability and RNA oxidation level of the pnp mutant cells in response to H2O2 treatment. Interestingly, degradosome-association is not required for PNPase to protect cell against oxidative stress. PNPase is evolutionary conserved in most of organisms of all domains of life. The human polynucleotide phosphorylase (hPNPase) localizes mainly in mitochondria and plays pleiotropic roles in cell differentiation and has been previously shown to bind 8- oxoG-RNA with a high affinity. Here we show that similar to E. coli PNPase, hPNPase plays an indispensable role in protecting HeLa cells against oxidative stress. The viability in HeLa cell and 8-oxoG levels in RNA are inversely correlated in response to H2O2- treatment. After removal of oxidative challenge, the elevated level of 8-oxoG in RNA decreases, suggesting the existence of surveillance mechanism(s) for cleaning up oxidized RNA. / We have shown that hPNPase may be responsible for the surveillance of oxidized RNA in mammalian cells.Overexpresion of hPNPase reduces RNA oxidation and increases HeLa cell viability against H2O2 insult. Conversely, hPNPase knockdown decreases the viability and increases 8-oxoG level in HeLa cells exposed to H2O2. Taken together, our results suggest that RNA oxidation is a challenging problem for living organisms, and PNPase may play an important role in protecting both prokaryotic and eukaryotic cells by limiting damage to RNA under oxidative stress. / by Jinhua Wu. / Thesis (Ph.D.)--Florida Atlantic University, 2008. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2008. Mode of access: World Wide Web.

Plusieurs données expérimentales suggèrent que la protéine Core du virus de l’Hépatite B (HBV), en plus de ses fonctions structurales pour la formation des nucléocapsides dans le cytoplasme, pourrait avoir des fonctions régulatrices importantes dans le noyau des hépatocytes infectés. En effet, Core s’associe à l’ADNccc et aux promoteurs de certains gènes cellulaires dans le noyau des hépatocytes infectés et pourrait ainsi contrôler leur régulation transcriptionnelle. De plus, de par sa capacité à lier les ARN, elle pourrait également participer au métabolisme post-transcriptionnel de gènes viraux et/ou cellulaires. Pour caractériser ces fonctions, nous avons réalisé une analyse protéomique des facteurs cellulaires qui interagissent avec la protéine Core dans le noyau d’hépatocytes humains. Cet interactome a mis en évidence un grand nombre de protéines de liaison aux ARN (RBP), qui participent au métabolisme des ARN et en particulier aux mécanismes d’épissage. Deux interactants majeurs de Core ont été plus particulièrement étudiés, SRSF10 et RBMX, impliqués notamment dans l’épissage et la réparation de l’ADN. Une analyse fonctionnelle effectuée par une approche siRNA a montré que SRSF10 et RBMX affectent différemment le niveau des ARN viraux, vraisemblablement en agissant à des étapes différentes du cycle viral. De même, un composé ciblant l’activité de certaines RBP diminue fortement la réplication d’HBV en affectant l’accumulation des ARN viraux. Ainsi, ces résultats suggèrent que Core pourrait interagir avec certaines RBP pour contrôler le destin des ARN viraux et/ou cellulaires, une piste intéressante pour le développement de nouvelles stratégies antivirales ciblant l’hôte / Converging evidences suggest that the Hepatitis B virus (HBV) core protein, beside its well-known structural role to form nucleocapsids in the cytoplasm, could have important regulatory functions in the nucleus of infected hepatocytes. Indeed, nuclear Core was shown to associate with the cccDNA and to the promoters of some cellular genes, suggesting that Core may control viral and/or cellular gene expression. In addition, Core has the capacity to bind RNA, and may thus regulate HBV RNA metabolism. To elucidate these functions, we performed a proteomic analysis of the cellular factors interacting with nuclear Core in human hepatocytes. This interactome revealed a majority of highly interconnected RNA-binding proteins (RBPs), which participate in several steps of mRNA metabolism, including transcription, splicing and nuclear egress. We focused on two major Core-interacting factors, SRSF10 and RBMX that were previously involved in splicing and DNA repair. Functional analyses performed by a siRNA approach indicated that RBMX and SRSF10 were able to differentially regulate the levels of all viral RNAs most likely by acting at different steps of the viral life-cycle. Similarly, a small compound, affecting the activity of selected RBPs, severely impaired HBV replication by strongly reducing viral RNA accumulation. Altogether, these results strongly suggest that Core interacts with some selected RBPs to control the fate of viral and/or cellular RNAs and provide new critical information for the development of novel host-targeting antiviral agents (HTA)