Browsing by Subject "DNA replication"
Now showing 1 - 4 of 4
Results Per Page
Sort Options
Item Isolation and genetic dissection of an eukaryotic replicon that supports autonomous DNA replication(Texas A&M University, 2007-04-25) Datta, ShibaniMaintenance of genome integrity requires that chromosomes be accurately and faithfully replicated. We are using Tetrahymena thermophila as a model system for studying the initiation and regulation of eukaryotic DNA replication. This organism contains a diploid micronucleus and polyploid macronucleus. During macronuclear development, the five diploid chromosomes of the micronucleus are fragmented into 280 macronuclear minichromosomes that are subsequently replicated to ~45 copies. In stark contrast, the 21 kb ribosomal DNA minichromosome (rDNA) is amplified from 2 to 10,000 copies in the same nucleus. Previous characterization of the rDNA replicon has led to the localization of its origin and the cis-acting regulatory determinants to the 1.9 kb 5'non-transcribed spacer region. The objective of this study was to identify and characterize non-rDNA origins of replication in Tetrahymena. This will help determine the underlying basis for differential regulation of rDNA and non-rDNA origins during development, as well as provide a better understanding of the organization of eukaryotic replicons. To this effect, I developed a DNA transformation assay that I used to isolate new Tetrahymena replication origins. A 6.7 kb non-rDNA fragment, designated TtARS1, was shown to support stable autonomous replication of circular plasmids in Tetrahymena. Genetic dissection revealed that TtARS1 contains two independent replicons, TtARS1-A and TtARS1-B. Full TtARS1-A function requires a minimal sequence of 700 bp, and two small regions in this fragment have been shown to be essential for origin function. TtARS1-B replicon function was localized to a 1.2 kb intergenic segment that contains little sequence similarity to TtARS1-A. Both non-rDNA replicons lack sequence similarity to the rDNA 5' NTS, suggesting that each replicon interact with a different set of regulatory proteins. This study indicates that the rDNA and the non-rDNA replicons have a modular organization, containing discrete, cis-acting replication determinants.Item Kinetics of DNA polymerase conformational changes during nucleotide binding and incorporation(2005) Tsai, Yu-chih; Johnson, Kenneth AllenItem Regulation of DNA Replication During Conventional and Unconventional Cell Cycles in Tetrahymena(2014-02-06) Lee, Po-HsuenAs the nucleating protein for pre-replicative complex (pre-RC) assembly, the conserved Origin Recognition Complex (ORC) specifies where replication initiates in eukaryotic chromosomes. During the vegetative cell cycle of Tetrahymena thermophila, previously published work has shown that DNA replication initiates from defined chromosomal sites in an ORC-dependent manner. Tetrahymena exhibits nuclear dimorphism, a polyploid somatic macronucleus (MAC), which is transcriptionally active and maintains vegetative growth, and a diploid germline micronucleus (MIC) responsible for the transmission of genetic information during conjugation. In order to provide more information about the fundamental mechanisms of micro- and macro- nuclear replication programs, I study the impacts of changing in ORC protein contents on the fate of micro- and macro- nuclear chromosomes during the vegetative cell cycle and development in Tetrahymena. I examined the effect of down-regulation of ORC1 on genome stability and intra- S phase checkpoint activation by disrupting ORC1 gene in the macronucleus. Partial depletion of Orc1p leads to genome instability in the diploid mitotic micronucleus, abnormal division of the polyploid amitotic macronucleus, and failure to mount a robust intra-S phase checkpoint response. In addition, the ORC1 knockdown strain fails to execute two developmentally- regulated DNA replication programs, endoreplication and ribosomal DNA (rDNA) gene amplification. I also examined the regulation of ORC and MCM during development. Remarkably, the result suggests that the demand on the ORC-dependent replication machinery differs during development and the vegetative S phase. To further gain new insights into fundamental mechanisms that protect chromosomes from replication stress, I examined the impact of replication stress on the regulation of ORC and MCM. This study led to the discovery of a novel DNA replication program that is activated under HU treatment. While Orc1p and Mcm6p were selectively degraded in response to HU, cells were competent to complete S phase in the absence of Orc1p and Mcm6p after HU was removed. In addition, the rDNA origin used exclusively during the S phase of vegetative cell cycle and developmentally programmed gene amplification is suppressed when these replication proteins are selectively degraded under HU treatment. Instead, an alternative program was used to resume the cell cycle progression. These data provide compelling evidence for an ORC-independent DNA replication program in cells recovering from replication stress.Item Structural and functional studies of the human mitochondrial DNA polymerase(2010-08) Lee, Young-Sam; Yin, Yuhui Whitney; Molineux, Ian J.; Johnson, Kenneth A.; Paull, Tanya T.; Robertus, Jon D.The human mitochondrial DNA polymerase (Pol γ) catalyzes mitochondrial DNA synthesis, and thus is essential for the integrity of the organelle. Mutations of Pol γ have been implicated in more than 150 human diseases. Reduced Pol γ activity caused by inhibition of anti-HIV drugs targeted to HIV reverse transcriptase confers major drug toxicity. To illustrate the structural basis for mtDNA replication and facilitate rational design of antiviral drugs, I have determined the crystal structure of human Pol γ holoenzyme. The structure reveals heterotrimer architecture of Pol γ holoenzyme with a monomeric catalytic subunit Pol γA, and a dimeric processivity factor Pol γB. While the polymerase and exonuclease domains in Pol γA present high structural homology with the other members of the DNA Pol I family, the spacer between the two functional domains shows a unique fold, and constitutes the subunit interface. The structure suggests a novel mechanism for Pol γ’s high processivity of DNA replication. Furthermore, the structure reveals dissimilarity in the active sites between Pol γ and HIV RT, thereby indicating an exploitable space for design of less toxic anti-HIV drugs. Interestingly, the structure shows an asymmetric subunit interaction, that is, one monomer of dimeric Pol γB primarily participates in interactions with Pol γA. To understand the roles of each Pol γB monomer, I generated a monomeric human Pol γB variant by disrupting the dimeric interface of the subunit. Comparative studies of this variant and dimeric wild-type Pol γB reveal that each monomer in the dimeric Pol γB makes a distinct contribution to processivity: one monomer (proximal to Pol γA) increases DNA binding affinity whereas the other monomer (distal to Pol γA) enhances the rate of polymerization. The pol γ holoenzyme structure also gives a rationale to establish the genotypic-phenotypic relationship of many disease-implicated mutations, especially for those located outside of the conserved pol or exo domains. Using the structure as a guide, I characterized a substitution of Pol γA residue R232 that is located at the subunit interface but far from either active sites. Kinetic analyses reveal that the mutation has no effect on intrinsic Pol γA activity, but shows functional defects in the holoenzyme, including decreased polymerase activity and increased exonuclease activity, as well as reduced discrimination between mismatched and corrected base pair. Results provide a molecular rationale for the Pol γA-R232 substitution mediated mitochondrial diseases.