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The effects of protein associations on pyrimidine deoxyribonucleotide biosynthesisMcGaughey, Kathleen M. 29 November 2001 (has links)
The faithful replication of DNA depends on the appropriate balance of DNA
precursors. From studies conducted in bacteriophage T4, models for
deoxyribonucleotide biosynthesis producing pools appropriate for DNA replication
have made it possible to understand more complex systems. A portion of that body
of evidence supports the concept that deoxyribonucleotide biosynthesis for
bacteriophage T4 is carried out by an association of enzymes and other cellular
components in a complex called the dNTP synthetase complex. This dissertation
explores potential direct protein-protein interactions within this complex for the
preparation of pyrimidine deoxyribonucleotides.
Direct associations for enzymes involved in pyrimidine deoxyribonucleotide
biosynthesis were examined by affinity chromatography. It was determined that there
was a significant direct relationship between T4 thymidylate synthase and T4 dCMP
deaminase, between T4 dCTPase/dUTPase and T4 dCMP deaminase as well. The
interaction between thymidylate synthase and dCMP deaminase was significantly
influenced by the presence of dCTP, a positive effector of dCMP deaminase.
Furthermore, protein associations changed the kinetic character of pyrimidine
deoxyribonucleotide production. T4 dCTPase/dUTPase, a member of the dNTP
synthetase complex, significantly alters the kinetic nature of thymidylate synthase by
working with thymidylate synthase in a reciprocal relationship. T4 single-stranded
DNA binding protein, a member of the replication complex, alters the activity of
thymidylate synthase as well. Attempts to isolate a kinetically coupled complex from
two or more constituent proteins of the dNTP synthetase complex were frustrated by
protein degradation to fragments under 10 kDa in size.
Pyrimidine deoxyribonucleotide synthesis is located between the significant
energy investment of ribonucleotide reductase and phosphate attachments by kinases
to prepare the deoxyribonucleotide molecules for DNA replication. In bacteriophage
T4, intermediate reactions are driven by mass action but are modulated by subtleties
including direct protein associations and the presence of small molecules that
influence enzyme function. Through these and potentially similar controls, pools of
deoxyribonucleotides are prepared and delivered in a timely, balanced manner to the
DNA replication apparatus. / Graduation date: 2002
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Precursors for mitochondrial DNA replication : metabolic sources and relations to mutagenesis and human diseasesSong, Shiwei 24 February 2005 (has links)
It is well known that the mitochondrial genome has a much higher spontaneous
mutation rate than the nuclear genome. mtDNA mutations have been identified in
association with many diseases and aging. mtDNA replication continues throughout the
cell cycle, even in post-mitotic cells. Therefore, a constant supply of nucleotides is
required for replication and maintenance of the mitochondrial genome. However, it is not
clear how dNTPs arise within mitochondria nor how mitochondrial dNTP pools are
regulated. Recent evidence suggests that abnormal mitochondrial nucleoside and
nucleotide metabolism is associated with several human diseases. Clearly, to uncover the
pathogenesis of these diseases and the mechanisms of mitochondrial mutagenesis,
information is needed regarding dNTP biosynthesis and maintenance within
mitochondria, and biochemical consequences of disordered mitochondrial dNTP
metabolism.
The studies described in this thesis provide important insight into these questions.
First, we found that a distinctive form of ribonucleotide reductase is associated with
mammalian liver mitochondria, indicating the presence of de novo pathway for dNTP
synthesis within mitochondria. Second, we found that long term thymidine treatment
could induce mtDNA deletions and the mitochondrial dNTP pool changes resulting from
thymidine treatment could account for the spectrum of mtDNA point mutations found in
Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) patients. These results
support the proposed pathogenesis of this disease. Third, we found that normal
intramitochondrial dNTP pools in rat tissues are highly asymmetric, and in vitro fidelity
studies show that these imbalanced pools can stimulate base substitution and frameshift
mutations, with a substitution pattern that correlates with mitochondrial substitution
mutations in vivo. These findings suggest that normal intramitochondrial dNTP pool
asymmetries could contribute to mitochondrial mutagenesis and mitochondrial diseases.
Last, Amish lethal microcephaly (MCPHA) has been proposed to be caused by
insufficient transport of dNTPs into mitochondria resulting from a loss-of-function
mutation in the gene encoding a mitochondrial deoxynucleotide carrier (DNC). We found
that there are no significant changes of intramitochondrial dNTP levels in both a MCPHA
patient's lymphoblasts with a missense point mutation in Dnc gene and the homozygous
mutant cells extracted from Dnc gene knockout mouse embryos. These results do not
support the proposed pathogenesis of this disease and indicate that the DNC protein does
not play a crucial role in the maintenance of intramitochondrial dNTP pools. / Graduation date: 2005
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Computational modeling of the hydrolysis of 2'-deoxyribonucleic acidsPrzybylski, Jennifer L., University of Lethbridge. Faculty of Arts and Science January 2009 (has links)
The mechanism for the hydrolysis of 2′-deoxyribonucleosides is examined using computational chemistry techniques. Initially, a model capable of accurately predicting the mechanism and activation barrier for the uncatalyzed hydrolysis of 2′-deoxyuridine is designed. It is found that the smallest model includes both explicit and implicit solvation during the optimization step. Next, this hybrid solvation model is applied to four natural nucleosides, namely 2′-deoxyadenosine, 2′-deoxycytidine, 2′-deoxyguanosine and thymidine. The hybrid model correctly predicts the trend in activation Gibbs energies for the pyrimidines and purines, separately. Finally, the concepts developed during the generation of the uncatalyzed hydrolysis model are applied to the mechanism of action of a glycosylase enzyme, namely human uracil DNA glycosylase. A hybrid ONIOM approach is utilized to study the experimentally proposed two-step mechanism. Results regarding the protonation state of His148 are inconclusive, and future directions are proposed. / xiii, [131] leaves : ill. (some col.) ; 29 cm
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Organization of the T4 dNTP synthetase complex at DNA replication sitesKim, JuHyun 02 February 2005 (has links)
With respect to a multienzyme complex of deoxyribonucleoside triphosphate
(dNTP) synthesis somehow juxtaposed with DNA replication sites, our laboratory
has demonstrated the existence of a multienzyme complex in T4-infected E. coli,
named the T4 dNTP synthetase complex, but the idea of direct linkage of dNTP
synthesis to DNA replication and organization of the complex has not been well
established. This study had two objectives. The first objective was to test the specific
hypothesis that gp32, the single-stranded DNA binding protein encoded by gene 32,
plays a role in recruiting enzymes of dNTP synthesis to the replisome and in
organizing the dNTP synthetase complex. By use of two newly created gene 32
mutants along with several experimental approaches, DNA-cellulose
chromatography, coimmunoprecipitation, and glutathione-S-transferase pull downs,
interactions of gp32 with thymidylate synthase (gptd), ribonucleotide reductase
(gpnrdA/B), and E. coli NDP kinase have been identified. These results support the
hypothesis that gp32 helps to recruit enzymes of dNTP synthesis to DNA replication
sites.
As the second objective, I investigated contributions of two host proteins, E. coli
nueleoside diphosphate kinase (NDP kinase) and adenylate kinase (Adk), to the
organization of the complex. As an important step to understand roles of E. coli NDP
kinase in the complex, I identified direct interactions of E. coli NDP kinase with
gpnrdA/B, dCMP hydroxymethylase (gp42), and dihydrofolate reductase (gpfrd) by
means of coimmunoprecipitation and glutathione-S-transferase pull-down
experiments. Interestingly, these interactions were influenced by the presence of
substrate nucleotides or an analog for E. coli NDP kinase, suggesting that metabolite
flux may affect the preference of E. coli NDP kinase binding to enzymes in the
complex in vivo. Meanwhile, Adk involvement in DNA precursor synthesis has been
suggested, particularly in phage T4-infected E. coli, from observations of increased
thermostability of temperature-sensitive Adk in situ. The involvement of E. coil Adk
in the complex was demonstrated by identifying some proteins of the T4 dNTP
synthetase complexgp42, dNMP kinase (gpl), gpfrd, and E. coli NDP
kinasedirectly interacting with Adk, implying that E. coil Adk would be properly
located in the complex to efficiently carry out the conversion of dNDPs to dNTPs.
This implication was supported by measurements of T4 DNA synthesis.
Taken together, this research strongly supports the idea of connection of dNTP
synthesis to DNA replication and allows us to take a step toward understanding the
organization of the complex at DNA replication sites. / Graduation date: 2005
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