Browsing by Subject "Biochemistry, Molecular Biology, and Biophysics"

Now showing 1 - 6 of 6
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    C-terminal flap endonuclease (rad27) mutations: lethal interactions with a DNA Ligase I mutation (cdc9-p) and suppression by proliferating cell nuclear antigen (POL30) in Saccharomyces cerevisiae.
    (2009-05) Karanja, Kenneth Kimani
    We recovered the flap endonuclease mutation rad27-K325* in a synthetic lethal screen with cdc9-p, a DNA ligase mutation with two substitutions (F43A/F44A) in its proliferating cell nuclear antigen (PCNA) interaction domain. We created two additional rad27 alleles, rad27-A358* with a stop codon at residue 358 and rad27-pX8 with substitutions of all eight residues of the PCNA interaction domain. Tests of mutation avoidance and DNA repair showed that rad27-K325* confers severe phenotypes similar to rad27Δ, rad27- A358* confers mild phenotypes and rad27-pX8 confers phenotypes intermediate between the other two alleles. rad27-K325* behaves similarly to rad27Δ in being lethal with exo1Δ and rad51Δ and not with rad2Δ. Interestingly, rad27-pX8 is lethal with rad51Δ, while rad27-A358* is lethal with rad51Δ at an elevated temperature. High copy expression of POL30 (PCNA) suppresses the canavanine mutation rate of all the rad27 alleles, including rad27Δ. rad27-K325* has an absolute lethality with the PCNA mutation pol30-90 that is not possessed by rad27Δ. These studies show the importance of the C-terminus of the flap endonuclease in mutation avoidance, and, by virtue of the initial screen, the role that PCNA plays in coordinating the entry of DNA ligase and the flap endonuclease in replication and repair.
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    The impact of loss of function mutations of NHEJ genes on gene targeting and DNA DSB repair in human somatic cells.
    (2009-04) Fattah, Farjana Jahan
    Non-homologous end-joining (NHEJ) is the predominant repair pathway for DNA double-strand breaks (DSBs) in human cells. The core NHEJ pathway is composed of seven factors: Ku70, Ku86, DNA-PKcs, Artemis, XRCC4, XLF and LIGIV. Mutation of any one of these NHEJ genes leads either to death, profound immune deficiencies, ionizing radiation sensitivity and/or cancer predisposition in human patients. We attempted to generate Ku70-null human somatic cells using a rAAV-based gene knockout strategy. Our data demonstrated that Ku70 is an essential gene in human somatic cells. More importantly, however, in Ku70+/- cells, the frequency of gene targeting was 5- to 10-fold higher than in wild type cells. RNA interference and short-hairpinned RNA strategies to deplete Ku70 phenocopied these results in wild-type cells and greatly accentuated them in Ku70+/- cell lines. Thus, Ku70 protein levels significantly influenced the frequency of rAAV-mediated gene targeting in human somatic cells. XLF is the newly identified core factor for NHEJ. To characterize XLF function in human cells, we knocked out XLF gene in HCT116 cells. XLF deficient cells are highly sensitive to ionizing radiation and DNA damaging agent, and have intrinsic DNA DSB repair defects. In V(D)J recombination assay, we find that XLF deficient cells have dramatic defect to form both V(D)J coding and signal joints. The phenotypes of XLF deficiency were rescued by a WT XLF cDNA over-expression. We conclude that, in humans, XLF is essential for C-NHEJ mediated repair of DNA-DSBs. Biochemical and genetic studies in mouse and hamster cells showed that DNA ends can also be joined via a backup pathway, especially when proteins responsible for NHEJ, are reduced or absent. In order to get insights in to backup NHEJ mechanism, we employed a reporter system based on the in vivo rejoining of cohesive and incompatible ends. We report here more than 10 to 20 fold reduction in NHEJ proficiency in DNA-PKcs, XLF and LIGIV null human cells, which is characterized by an increase in microhomology use. Strikingly, conditional knock-out of Ku86 did not result in defect in end-joining, while having an impact on repair fidelity.
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    Molecular analysis of components in Drosophila polycomb group silencing.
    (2009-04) Jahren, Neal Richard
    The aim of this research is to describe the molecular mechanism used by a set of regulatory proteins, called Polycomb Group proteins, to silence genes. In general, the function of the Polycomb Group is to silence cell differentiation factors in the course of embryonic development by operating on chromatin. First, we investigate a recently discovered Polycomb protein to determine its expression profile during development and to establish its role within a complex of proteins that methylates chromatin during Polycomb repression. Second, we target the methyltransferase complex to artificial loci on chromosomes by fusing one of its subunits to a heterologous DNA-binding domain. Third, we investigate the recruitment of the SCM Polycomb Group protein to chromatin during silencing. We perform molecular epistasis experiments to determine the dependencies between SCM and the other Polycomb Group proteins for arriving at chromatin. These studies use the Drosophila model system. The experiments include the observation of phenotypes resulting from genetic crosses and modifications, immunostaining of tissues and chromosomes, and chromatin immunoprecipitation. The Polycomb Group of proteins is highly conserved from Drosophila to humans, so the results of this research should yield insights about gene regulation in all higher organisms.
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    PFOA alters the expression of mitochondrial metabolism genes in rats.
    (2010-06) Walters, Mark William
    Abstract summary not available.
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    Protein-protein interactions and the characterization of adipocyte fatty acid binding protein.
    (2009-01) Thompson, Brian Raymond
    Adipose tissue functions to not only store and provide an energy source in the form of triacylglycerol but to signal the nutritional status of those reserves through the secretion of adipokines and lipids. In obesity, this homeostatic regulation of whole body lipid metabolism is compromised resulting in disease states, such as type 2 diabetes and atherosclerosis. Adipocyte fatty acid binding protein, AFABP/aP2, functions to solubilize and traffic fatty acids, and other lipid metabolites, within the aqueous environment of the adipocyte. AFABP/aP2 knockout mice are resistance to diet and genetic induced obesity linked insulin resistance, while still being obese. The molecular mechanisms of this uncoupling of obesity and insulin resistance have not been elucidated. The identification and characterization of HSL and JAK2 as interacting partners of AFABP/aP2 are presented here. Fatty acid binding by AFABP/aP2 is necessary for the interactions, as indicated by the loss of interactions with the fatty acid binding mutant R126L/Y128F of AFABP/aP2. The phosphorylation state of HSL and JAK2 regulates their interaction with AFABP/aP2. An ExYK motif in AFABP/aP2, DDYMK, in the helix-turn-helix domain is the site of these interactions and a similar ExYK motif in HSL and JAK2 are important for these interactions. We propose that AFABP/aP2 is a fatty acid sensor, through lipid-regulated interaction, from AFABP/aP2's helix-turn-helix domain to an ExYK motif in HSL and JAK2. Furthermore, through its lipid-regulated interactions, AFABP/aP2 is a homeostatic regulator of whole body lipid metabolism, coordinating gene expression, signal transduction and metabolism.
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    Structure-Activity Relationships Between Perfluorinated Chemicals and Mitochondrial Respiration Rates
    (2010-02) Ray, Josiah Nathanael
    Perfluorinated chemicals are synthesized compounds that are structurally derived from hydrocarbons, but have carbon-hydrogen bonds replaced with carbon-fluorine bonds. They have been shown to inhibit respiration in rat liver mitochondria in vitro. The objective of this investigation was to explore whether structure-activity relationships exist for the effects of perfluorinated chemicals on mitochondrial respiration. Freshly isolated rat liver mitochondria were incubated with increasing concentrations of one of fifteen different perfluorinated chemicals, after which oxidative phosphorylation was recorded polarographically with an oxygen electrode. Each analysis was performed with five concentrations of the respective perfluorinated chemical, and repeated with mitochondria isolated from five individual animals. The respiration profiles of structurally related perfluorinated chemicals were then compared to each other. For some perfluorinated chemicals, increased carbon-chain length corresponded with an increased impact on mitochondrial respiration. However, it appeared that the effect of chain length was secondary to the ability of the perfluorinated chemical to efficiently carry protons across the mitochondrial membrane.