The Metnase SET domain is necessary for Metnase function in damage recovery prior to replication restart following HU treatment.

<p>Representative images of γ-H2AX and RPA p70 foci in HEK293 cells stably transfected with pCMV4 vector, wt-Metnase, and the SET deletion mutant (Δ<i>all</i>-SET), as indicated on the top. After treatment with 2 mM HU for 3 hrs, cells were released into fresh media at indicated times, stained with DAPI (blue) and antibodies to γ-H2AX (green) and RPA p70 (red), and imaged by confocal microscopy.</p

The Metnase SET domain is necessary for the 5’-end cleavage of ss-overhang DNA.

<p>(A) Silver staining of purified wt-Metnase (<i>wt-</i>MET), the SET deletion mutant (Δ<i>all</i>-SET), and a nuclease-dead mutant (D483A) following 10% SDS-PAGE. (B) The SET domain is essential for cleavage of the 5’-flap DNA. Reaction mixtures (20 μl) containing the 5’-³²P-labeled flap DNA (60 fmol) and increasing amounts of <i>wt</i>-Metnase or the mutant (Δ<i>all</i>-SET or D483A) were incubated at 37°C in the presence of 2 mM MgCl₂ for 90 min, and cleavage products were analyzed by 12% PAGE containing 8M urea. Numbers on the left indicated DNA size makers. (C-D) Cleavage of the branch site (panel C) and the 5’ end of ss-overhang (panel D) of a 5’-flap DNA shown in Panel B was quantified using a PhosphorImager and ImageQuant software (Molecular Dynamics). (E) Cleavage of the 5’ end of the 5’-³²P-labeled partial duplex DNA with wt-Metnase and the SET deletion mutant. Indicated amount of wt-Metnase or the Δ<i>all</i>-SET was incubated with the 5’-³²P-labeled (*) 5’-ss-overhang DNA (60 fmol) for 90 min at 37°C prior to 12% PAGE analysis (+ 8M urea). Arrows on the right mark the cleavage sites on the 5’-³²P-labeled (*) DNA.</p

The Metnase SET domain is necessary for replication recovery after long exposure to hydroxyurea (HU).

<p>(A) Schematic diagram of Metnase. The Metnase SET domain comprises pre-SET (aa 14–118), SET (aa 119–260), and post-SET (aa 261–326) domains. The <i>pre</i> SET domain contains a cysteine-rich putative Zn⁺² binding motif (10 Cys), while the SET domain has the SAM binding motif (207-RFLNHxCxPN----ELxYDY-248) for histone lysine methyltransferase activity. The <i>post</i>-SET domain also contains a cysteine-rich putative Zn⁺² binding motif (CxCx₄C). (B) Western blot analysis of flag-tagged wt-Metnase and the SET mutants that were stably expressed in HEK293 cells. Thirty μg of cell extracts were loaded onto 10% SDS-PAGE for immunoblot analysis. Ku80 was used as a loading control. (C) Metnase expression in control HEK293 cells (mock) and wt-Metnase overexpressor (wt-Met⁺) was analyzed by RT-PCR. (D) Representative confocal microscope images of HEK293 cells stably transfected with pCMV4 vector (top row), wt-Metnase (second row), and the SET mutants (third to sixth row) following HU treatment. After treatment of 2 mM HU for 3 hrs, cells were released into fresh media at indicated times, stained with DAPI (blue) and an antibody to γ-H2AX (green), and imaged by confocal microscopy. (E) Quantitation of γ-H2AX-positive cells in panel D. Plots were average percentages (±SD) of γ-H2AX-positive cells. An average of 200 cells were counted per slide, 6 slides per experiment. **, P<0.01; ***, P<0.005, t tests.</p

The Metnase SET domain is not involved in the Metnase-DNA interaction.

<p><b>(A)</b> Indicated amounts of wt-Metnase or the Δ<i>all</i>-SET mutant were incubated with 400 fmol of 5’-³²P-labeled TIR DNA. Following 15 min incubation at 25°C, the protein–DNA complexes were analyzed by 5% native PAGE in the presence of 1X TBE. (B-C) Quantitation of free DNA and the Metnase-TIR complex. For quantitation, individual bands were excised from dried gel and measured for radioactivity. (D-F) Interaction of wt-Metnase and the SET deletion mutant (Δ<i>all</i>-SET) with DNA using Streptavidine pulldown assay. Flag-tagged <i>wt</i>-Metnase or Δ<i>all</i>-SET protein (1.0 and 2.0 μg) was incubated with 50 pmol of the 3’-biotinylated 5’-flap DNA (panel D), a partial duplex DNA (panel E), or ssDNA (panel F) for protein-DNA binding by Streptavidin-agarose beads (see Experimental Procedures for the details). The protein-DNA interaction was analyzed by Western blot using an anti-flag antibody. (G) Salt sensitivity of wt-Metnase and the SET deletion mutants in their interaction with a 5’-flap DNA. Wt-Metnase or the SET deletion mutant (2.0 μg) was incubated with 50 pmol of the 3’-biotinylated 5’-flap DNA in the presence of varying concentrations of NaCl prior to DNA pull-down with streptavidin-agarose beads. The protein-DNA interaction was analyzed by Western blot using an anti-flag antibody. (H) The protein-DNA complexes (panel G) were quantified by Molecular Imager ChemiDoc XRS using Quantity One^<b>®</b> analysis software program (BioRad).</p

The Metnase SET domain but not its HLMT activity is essential for cleavage of the 5’ end of a 5’-flap DNA.

<p>(A) Silver staining of purified wt-Metnase (<i>wt-</i>MET) and the SET deletion and the substitution mutants following 10% SDS-PAGE. (B) Cleavage of the 5’ end of a 5’-flap DNA with the SET deletion and the substitution mutants. Increasing amounts of <i>wt</i>-Metnase and the SET domain deletion mutant (panel B) were incubated with 60 fmol of a 5’-³²P-flap DNA for 120 min prior to 12% denatured PAGE (+ 8 M urea) analysis. (C-D) Metnase-mediated cleavage of the branch site (panel C) and the 5’ end of ss-overhang (panel D) of a 5’-flap DNA shown in Panel B was quantified. (E) Increasing amounts of <i>wt</i>-Metnase and the substitution mutants lacking HLMT activity (N210S & D248S) were examined for cleavage of the 5’-flap DNA.</p

Facile Route to the Controlled Synthesis of Tetragonal and Orthorhombic SnO₂ Films by Mist Chemical Vapor Deposition

Two types of tin dioxide (SnO₂) films were grown by mist chemical vapor deposition (Mist-CVD), and their electrical properties were studied. A tetragonal phase is obtained when methanol is used as the solvent, while an orthorhombic structure is formed with acetone. The two phases of SnO₂ exhibit different electrical properties. Tetragonal SnO₂ behaves as a semiconductor, and thin-film transistors (TFTs) incorporating this material as the active layer exhibit n-type characteristics with typical field-effect mobility (μ_FE) values of approximately 3–4 cm²/(V s). On the other hand, orthorhombic SnO₂ is found to behave as a metal-like transparent conductive oxide. Density functional theory calculations reveal that orthorhombic SnO₂ is more stable under oxygen-rich conditions, which correlates well with the experimentally observed solvent effects. The present study paves the way for the controlled synthesis of functional materials by atmospheric pressure growth techniques

Additional file 1:

Case form and result of data. (XLSX 31 kb

High-Performance Zinc Tin Oxide Semiconductor Grown by Atmospheric-Pressure Mist-CVD and the Associated Thin-Film Transistor Properties

Zinc tin oxide (Zn–Sn–O, or ZTO) semiconductor layers were synthesized based on solution processes, of which one type involves the conventional spin coating method and the other is grown by mist chemical vapor deposition (mist-CVD). Liquid precursor solutions are used in each case, with tin chloride and zinc chloride (1:1) as solutes in solvent mixtures of acetone and deionized water. Mist-CVD ZTO films are mostly polycrystalline, while those synthesized by spin-coating are amorphous. Thin-film transistors based on mist-CVD ZTO active layers exhibit excellent electron transport properties with a saturation mobility of 14.6 cm²/(V s), which is superior to that of their spin-coated counterparts (6.88 cm²/(V s)). X-ray photoelectron spectroscopy (XPS) analyses suggest that the mist-CVD ZTO films contain relatively small amounts of oxygen vacancies and, hence, lower free-carrier concentrations. The enhanced electron mobility of mist-CVD ZTO is therefore anticipated to be associated with the electronic band structure, which is examined by X-ray absorption near-edge structure (XANES) analyses, rather than the density of electron carriers

Additional file 3: Figure S3. of The endonuclease EEPD1 mediates synthetic lethality in RAD52-depleted BRCA1 mutant breast cancer cells

EEPD1 depletion in BRCA1-depleted MCF7 breast cancer cells rescues synthetic lethality from RAD52 depletion. a–c MCF7 BRCA1-proficient cells were transiently transfected with control or RAD52 siRNA, with or without BRCA1 siRNA, for 48 h. Cells were plated for colony formation survival assays. a Western blot analysis. b Representation images of CFUs from each condition after 14 days. c Quantitative analysis of colony formation. d–f MCF7 BRCA1-proficient cells were transiently transfected with control, EEPD1 and/or RAD52 siRNA, with BRCA1 siRNA, for 48 h. Cells were plated for colony formation survival assays. d Western blot analysis. e Representation images of CFUs from each condition after 14 days. f Quantitative analysis of colony formation. Each experiment was performed ≥ 3 distinct times in triplicate. (PDF 459 kb

Additional file 4: Figure S4. of The endonuclease EEPD1 mediates synthetic lethality in RAD52-depleted BRCA1 mutant breast cancer cells

Single-label DNA fiber analysis of stressed replication fork degradation. MDA-MB-436 BRCA1-/- cells were transiently transfected with control, EEPD1 and/or RAD52 siRNA for 48 h and labeled with IdU for 45 min before proceeding to either 0 h or 10 h incubation with 5 mM HU. DNA degradation at stalled nascent replication forks was measured by fiber analysis. a Schematic diagram depicts steps for the DNA fiber assay and representative images of DNA fibers from each condition. IdU stained red (stalled forks). Quantitative analysis of IdU track length frequency at unstressed (no HU) (b), or HU-treated DNA fibers (c) from each condition. d Bar chart from the HU-treated IdU track length frequencies analysis. c and d are the same data. Co-depletion of both RAD52 and EEPD1 restores stressed replication fork degradation back to control levels. Three distinct experiments per condition (>100 fibers measured per condition for each experiment). (PDF 419 kb