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No description available.

Methodology was developed which allowed the rapid and routine quantitation of subpicomole quantities of diadenosine-5ʹ,5ʹʹʹ-P¹,P⁴-tetraphosphate (Ap₄A) in cultured mammalian cells. This methodology includes the rapid extraction of cellular nucleotides in cold alkali, resolution of Ap₄A from the bulk of cellular materials on a highly specific boronate affinity resin, and quantitation of the dinucleotide in a coupled bioluminescence assay utilizing venom phosphodiesterase and firefly luciferase. The sensitivity and selectivity of this assay is demonstrated and contrasted with previously developed techniques. This assay was used to examine the role of Ap₄A in DNA replication and the cellular stress response.

H1 and core histone mRNA levels have been examined in the presence of portein synthesis inhibitors with different mechanisms of action. Total HeLa cell RNAs were analyzed by Northern Blot hybridization using cloned human histone genes as probes. Inhibition of DNA replication resulted in a rapid decline in histone mRNA levels. However, in the presence of cycloheximide or puromycin, H1 and core mRNAs did not decrease in parallel with DNA synthesis, but were stabilized and accumulated. Inhibition of DNA synthesis with hydroxyurea after the inhibition of protein synthesis did not lead to a decline in histone mRNA levels. These results suggest that synthesis of a protein(s) - perhaps a histone protein(s) - is required for the coordination of DNA synthesis and histone mRNA levels.

Indiana University-Purdue University Indianapolis (IUPUI) / DNA suffers constant damage, leading to a variety of lesions that require repair. One of the most devastating lesions is a double-strand break (DSB), which results in physical dissociation of two pieces of a chromosome. Necessarily, cells have evolved a number of DSB repair mechanisms. One mechanism of DSB repair is break-induced replication (BIR), which involves invasion of one side of the broken chromosome into a homologous template, followed by copying of the donor molecule through telomeric sequences. BIR is an important cellular process implicated in the restart of collapsed replication forks, as well as in various chromosomal instabilities. Furthermore, BIR uniquely combines processive replication involving a replication fork with DSB repair. This work employs a system in Saccharomyces cerevisiae to investigate genetic control, physical outcomes, and frameshift mutagenesis associated with BIR initiated by a controlled HO-endonuclease break in a chromosome. Mutations in POL32, which encodes a third, non-essential subunit of polymerase delta (Pol delta), as well as RAD9 and RAD24, which participate in the DNA damage checkpoint response, resulted in a BIR defect characterized by decreased BIR repair and increased loss of the broken chromosome. Also, increased incidence of chromosomal fusions determined to be half-crossover (HCO) molecules was confirmed in pol32 and rad24, as well as a rad9rad50S double mutant. HCO formation was also stimulated by addition of a replication-inhibiting drug, methyl-methane sulfonate (MMS), to cells undergoing BIR repair. Based on these data, it is proposed that interruption of BIR after it has initiated is one mechanism of HCO formation. Addition of a frameshift mutation reporter to this system allowed mutagenesis associated with BIR DNA synthesis to be measured. It is demonstrated that BIR DNA synthesis is intrinsically inaccurate over the entire path of the replication fork, as the rate of frameshift mutagenesis during BIR is up to 2800-fold higher than normal replication. Importantly, this high rate of mutagenesis was observed not only close to the DSB where BIR is less stable, but also far from the DSB where the BIR replication fork is fast and stabilized. Pol  proofreading and mismatch repair (MMR) are confirmed to correct BIR errors. Based on these data, it is proposed that a high level of DNA polymerase errors that is not fully compensated by error-correction mechanisms is largely responsible for mutagenesis during BIR. Pif1p, a helicase that is non-essential for DNA replication, and elevated dNTP levels during BIR also contributed to BIR mutagenesis. Taken together, this work characterizes BIR as an essential repair process that also poses risks to a cell, including genome destabilization and hypermutagenesis.

No description available.

Plants have two organelles outside the nucleus which carry their own DNA, mitochondria and chloroplasts. These organelles are descendants of bacteria that were engulfed by their host according to the endosymbiotic theory. Over time, DNA has been exchanged between these organelles and the nucleus. Two polymerases, DNA Polymerases Gamma I and II, are encoded in the nucleus and remain under nuclear control, but are transported into the mitochondria and chloroplasts. DNA polymerases gamma I and II are two organelle polymerases which have been studied through sequence analysis and shown to localize to both mitochondria and chloroplasts. Little has been done to characterize the activities of these polymerases. Work in tobacco showed the homology of these polymerases to each other and to DNA Polymerase I in bacteria. They have been characterized as being part of the DNA Polymerase A family of polymerases. In my research I have studied the effect of T-DNA insertions within the DNA Polymerase Gamma I and II genes. Since these DNA Polymerases are targeted to the mitochondria and chloroplasts, I studied the effect of knocking out these genes. A plant heterozygous for an insert in DNA Polymerase Gamma I grows slightly slower than wild type plants with an approximately 20% reduction in mitochondrial and chloroplast DNA copy number. A plant homozygous for an insert in this same gene has a drastic phenotype with stunted plants that grow to around 1 inch tall, with floral stems, and have an approximately 50-55% reduction in mitochondrial and chloroplast DNA copy number. Wild type plants can grow to a height of 12-18 inches with floral stems as a comparison. A plant heterozygous for an insert in the DNA Polymerase Gamma II gene grows slightly slower than wild type plants and has an approximately 15% reduction in mitochondrial DNA copy number and a 50% reduction in chloroplast DNA copy number. These plants also produce much less seed than do other mutants and wild type plants.

Nucleic acids are complex macromolecules that are susceptible to both endogenous and exogenous damage. This study explored damage resulting from interactions with environmental nucleophilic toxins, such as a variety of diols and amines found in industry. These nucleophiles can react with electrophilic groups, such as 5-formylcytosine. 5-formylcytosine is an oxidation product of the epigenetic base 5-methylcytosine. It is typically removed by thymine DNA glycosylase (TDG) but is known to accumulate in the genome, making the formyl group susceptible to attack. In this study we used GC/MS and ESI-MS to show that DNA lesions from the nucleophilic addition reaction of toxins and 5-formylcytosine can be formed under physiological conditions. In addition, this formation showed a pH dependency, with lower pHs showing more product formation. Studies with a lesion formed from the reaction of 1,3-propane diol and 5-formylcytosine showed that the lesion has little effect on the conformation of the DNA duplex. UV thermal denaturation studies showed that at a glance the lesion also has little effect on the stability of the DNA duplex, however, more extensive studies revealed a slight destabilization effect due to the lesion. Enzymatic studies showed that the presence of one lesion does not have a significant effect on the ability of DNA polymerase to efficiently complete DNA replication with high fidelity, but when the lesion was incorrectly base paired, the extension reactions resulted in deletion products or a halt in replication. Addition of a second tandem lesion to the template resulted in a decrease in fidelity, while continuing to give deletion products and replication stops in the presence of mismatched base pairs. This is particularly significant, indicating the potential for the lesion to be mutagenic or even cytotoxic. Lesions formed from other environmental toxins could be even more damaging, making them well worth future investigation.

DNA needs to be accurately copied during DNA replication for a normal cell function. Errors during DNA replication can cause genomic instability that can lead to cancer. To avoid mistakes during the process of DNA replication, nuclease enzymes can act as molecular scissors in removing lethal DNA structures. Therefore, Flap endonuclease 1 (FEN1) is an enzyme that can cleave the 5’flap primer during Okazaki fragment maturation. However, studies have shown that overexpression of FEN1 is associated with different types of cancer. Thus, targeting FEN1 represents a potential for enhancing cancer therapy. However, structural investigation of FEN1 and factors that influence DNA binding need to be comprehensively studied at the molecular level before designing an inhibitor. Thus, this thesis aimed to investigate and compare the catalytic behavior of FEN1wt, FEN1K93A, and FEN1D181A in different experimental conditions. We have found that the activity of FEN1 is affected by the presence of divalent metal ions such as Ca2+ and Mg2+ by performing enzymatic assays. Using the microscale thermophoresis technique, we determined the dissociation constants for FEN1 proteins. Additionally, we performed a thermal shift assay in different conditions which gave us additional insights into the stability of the protein-DNA complex in FEN1. We have found that protein-DNA complex in FEN1D181 is more stable than FEN1wt and FEN1K93A by having a higher melting temperature. Lastly, I used the NMR technique to map the conformational changes within FEN1 proteins upon interacting with divalent metal ions such as Mg2+ ions. To do this, I performed a series of Mg2+ ions titration for FEN1 (WT, K93A, and D181A) using a 2D 1 H 15N TROSY-HSQC experiment to monitor the chemical shifts changes to the chemical environment around the N-H backbone amides of the protein. We have found that both WT and K93A FEN1 proteins interact in a similar way with Mg2+ ions, i.e., explicitly targeting first the higher affinity catalytic site, then spreading around several unspecific low-affinity sites across the protein; however, we observed only the unspecific and weak milli molar binding affinity in FEN1D181A across the entire protein surface upon interacting with Mg2+ ions.

京都大学 / 新制・課程博士 / 博士(エネルギー科学) / 甲第24326号 / エネ博第454号 / 新制||エネ||85(附属図書館) / 京都大学大学院エネルギー科学研究科エネルギー基礎科学専攻 / (主査)教授 片平 正人, 教授 森井 孝, 教授 杤尾 豪人 / 学位規則第4条第1項該当 / Doctor of Energy Science / Kyoto University / DGAM