11 research outputs found

    The time evolution of 〈<i>C</i><sub><i>n</i></sub>〉.

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    <p>Time evolution of 〈<i>C</i><sub><i>n</i></sub>〉 corresponding to the three cases in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132397#pone.0132397.g002" target="_blank">Fig 2(A), 2(B) and 2(C)</a> shown in solid, dotted and dashed lines, respectively.</p

    High- and low-<i>β</i> limits for ⟨<i>C</i><sub><i>n</i></sub>⟩.

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    <p>Various trajectories for ⟨<i>C</i><sub><i>n</i></sub>⟩ for increasing <i>ϵ</i>/<i>η</i> plotted for various <i>β</i> at <i>γ</i> = 0.5. Shown are <i>β</i> values where <i>β</i> = 10<sup><i>n</i></sup> (<i>n</i> = −0.5, 0, 0.5, …, 2.0). Trajectories of ⟨<i>C</i><sub><i>n</i></sub>⟩ approach a limit as <i>β</i> increases from small <i>β</i> = 10<sup>−0.5</sup> (long dashes) to large <i>β</i> = 10<sup>2</sup> (right-most solid).</p

    Biochemical Characterization of Eight Genetic Variants of Human DNA Polymerase κ Involved in Error-Free Bypass across Bulky <i>N</i><sup>2</sup>‑Guanyl DNA Adducts

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    DNA polymerase (pol) κ, one of the Y-family polymerases, has been shown to function in error-free translesion DNA synthesis (TLS) opposite the bulky <i>N</i><sup>2</sup>-guanyl DNA lesions induced by many carcinogens such as polycyclic aromatic hydrocarbons. We analyzed the biochemical properties of eight reported human pol κ variants positioned in the polymerase core domain, using the recombinant pol κ (residues 1–526) protein and the DNA template containing an <i>N</i><sup>2</sup>-CH<sub>2</sub>(9-anthracenyl)­G (<i>N</i><sup>2</sup>-AnthG). The truncation R219X was devoid of polymerase activity, and the E419G and Y432S variants showed much lower polymerase activity than wild-type pol κ. In steady-state kinetic analyses, E419G and Y432S displayed 20- to 34-fold decreases in <i>k</i><sub>cat</sub>/<i>K</i><sub>m</sub> for dCTP insertion opposite G and <i>N</i><sup>2</sup>-AnthG compared to that of wild-type pol κ. The L21F, I39T, and D189G variants, as well as E419G and Y432S, displayed 6- to 22-fold decreases in <i>k</i><sub>cat</sub>/<i>K</i><sub>m</sub> for next-base extension from C paired with <i>N</i><sup>2</sup>-AnthG, compared to that of wild-type pol κ. The defective Y432S variant had 4- to 5-fold lower DNA-binding affinity than wild-type, while a slightly more efficient S423R variant possessed 2- to 3-fold higher DNA-binding affinity. These results suggest that R219X abolishes and the E419G, Y432S, L21F, I39T, and D189G variations substantially impair the TLS ability of pol κ opposite bulky <i>N</i><sup>2</sup>-G lesions in the insertion step opposite the lesion and/or the subsequent extension step, raising the possibility that certain nonsynonymous pol κ genetic variations translate into individual differences in susceptibility to genotoxic carcinogens

    Biochemical Characterization of Eight Genetic Variants of Human DNA Polymerase κ Involved in Error-Free Bypass across Bulky <i>N</i><sup>2</sup>‑Guanyl DNA Adducts

    No full text
    DNA polymerase (pol) κ, one of the Y-family polymerases, has been shown to function in error-free translesion DNA synthesis (TLS) opposite the bulky <i>N</i><sup>2</sup>-guanyl DNA lesions induced by many carcinogens such as polycyclic aromatic hydrocarbons. We analyzed the biochemical properties of eight reported human pol κ variants positioned in the polymerase core domain, using the recombinant pol κ (residues 1–526) protein and the DNA template containing an <i>N</i><sup>2</sup>-CH<sub>2</sub>(9-anthracenyl)­G (<i>N</i><sup>2</sup>-AnthG). The truncation R219X was devoid of polymerase activity, and the E419G and Y432S variants showed much lower polymerase activity than wild-type pol κ. In steady-state kinetic analyses, E419G and Y432S displayed 20- to 34-fold decreases in <i>k</i><sub>cat</sub>/<i>K</i><sub>m</sub> for dCTP insertion opposite G and <i>N</i><sup>2</sup>-AnthG compared to that of wild-type pol κ. The L21F, I39T, and D189G variants, as well as E419G and Y432S, displayed 6- to 22-fold decreases in <i>k</i><sub>cat</sub>/<i>K</i><sub>m</sub> for next-base extension from C paired with <i>N</i><sup>2</sup>-AnthG, compared to that of wild-type pol κ. The defective Y432S variant had 4- to 5-fold lower DNA-binding affinity than wild-type, while a slightly more efficient S423R variant possessed 2- to 3-fold higher DNA-binding affinity. These results suggest that R219X abolishes and the E419G, Y432S, L21F, I39T, and D189G variations substantially impair the TLS ability of pol κ opposite bulky <i>N</i><sup>2</sup>-G lesions in the insertion step opposite the lesion and/or the subsequent extension step, raising the possibility that certain nonsynonymous pol κ genetic variations translate into individual differences in susceptibility to genotoxic carcinogens

    Hypoxia induces the ERRγ gene expression in hepatoma cell lines.

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    <p>(<b>A–B</b>), HepG2 cells were seeded in 60-cm<sup>2</sup> dishes and incubated overnight. Then cells were incubated under hypoxia at indicated time period. The expression of ERRγ was analyzed by Western blot (<b>A</b>) and Q-PCR (<b>B</b>) analysis. (<b>C–D</b>) HepG2 cells were seeded in 60-cm<sup>2</sup> dishes and incubated overnight and then treated with DFO at indicated concentration and time period. The expression of ERRs was analyzed by Western blot (<b>C</b>) and Q-PCR (<b>D</b>) analysis. ERRγ gene expression was normalized to L32 gene expression, and α or β-tubulin expression. All data are representative of at least three independent experiments. Error bars show ± S.E.M. <sup>*</sup><i>P</i><0.05, <sup>**</sup><i>P</i><0.01 by two-tailed Student <i>t</i>-test.</p

    ERRγ directly regulates hypoxia mediated PDK4 gene expression.

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    <p>(<b>A</b>) HepG2 cells were transfected with hPDK4-Luc. After transfection, the cells were exposed to hypoxia for indicated period and lysates were utilized for luciferase and β-galactosidase assay. Experiments were done in triplicate and data are expressed as the fold activation relative to the control. (<b>B</b>) HepG2 cells were seeded in 60-cm<sup>2</sup> dishes and trasnfected siERRγ and control siRNA for 72 hr and then exposed hypoxia for 9 hr. Cells were harvested for analyzing luciferase and β-galactodidase assay. (<b>C–D</b>) HepG2 cells were transfected with several deletion constructs of hPDK4 (−848)-Luc, hPDK4 (−500)-Luc, hPDK4 (−291)-Luc and hPDK4-mtERRE1-Luc with pcDNA3-ERRγ in the presence or absence with hypoxia exposure, respectively. 48 hr after transfection, the cells were harvested and performed luciferase and β-galactodidase assay. Experiments were done in duplicate and data expressed as the fold activation related to control. (<b>E</b>) HepG2 cell were transiently transfected with hPDK4 (−848)-Luc, hPDK4-mtERRE1-Luc, pcDNA3-ERRγ and pcDNA3-HIF-1α. (<b>F</b>) ChIP assay: HepG2 cell was exposed to hypoxia for 9 hr. Input represents 10% of purified DNA in each sample. Cell extracts were immunoprecipitated with anti-ERRα and purified DNA samples were employed for Q-PCR with primers binding to ERRE (−502 to −252) and distal site (−1056 to −886) on the <i>PDK4</i> gene promoter. All data are representative of at least three independent experiments. Error bars show ± S.E.M. <sup>***</sup><i>P</i><0.001 by two-tailed Student <i>t</i>-test.</p
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