ATPase assays were performed as described before (6 (link)). In brief, MipZ-His6 or its variants (6 μM) were preincubated for 10 min at 30°C in buffer P (50 mM HEPES/NaOH pH 7.2, 50 mM KCl, 10 mM MgCl2, 1 mM β-mercaptoethanol). The reaction was started by the addition of 1 mM ATP containing [α-32P]ATP (25 Ci/mmol) (Hartmann, Germany). Samples (2 μl) were taken every 10 min over a period of 1 h and transferred onto PEI-cellulose F thin-layer chromatography plates (Merck, Germany). The plates were developed in a solvent system containing 1 M LiCl and 0.5 M formic acid, air-dried, and exposed to a phosphor screen (GE Healthcare, USA). After scanning of the screen in a Storm 840 PhosphorImager (GE Healthcare, Germany), the amount of [α-32P]ADP in the samples was quantified using ImageQuant 5.2 (GE Healthcare). The reaction rates were determined by linear-regression analysis in Microsoft Excel 2010.
Storm 840 phosphorimager
Manufactured by GE Healthcare
Sourced in Germany, United States
The Storm 840 PhosphorImager is a laboratory instrument designed for the detection and quantification of radiolabeled molecules, such as proteins, nucleic acids, and other biomolecules. It utilizes phosphor imaging technology to capture and digitize images of radioactive samples, providing a sensitive and efficient method for analyzing experimental results.
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Lab products found in correlation
Imagequant 5, by GE Healthcare (9 mentions)
Whatman, by Cytiva (5 mentions)
Nylon membrane, by Cytiva (4 mentions)
Hybond, by Cytiva (3 mentions)
Imagequant software, by GE Healthcare (3 mentions)
De81 chromatography paper, by Cytiva (3 mentions)
Masterpure yeast rna purification kit, by Illumina (2 mentions)
Nucleotrap mrna purification kit, by Takara Bio (2 mentions)
Deae 81 paper, by Cytiva (2 mentions)
Phosphor screen, by GE Healthcare (1 mentions)
Sybr green 1, by Thermo Fisher Scientific (1 mentions)
Protein g coupled dynabeads, by Thermo Fisher Scientific (1 mentions)
Anti flag m2, by Merck Group (1 mentions)
Hybond xl membrane, by GE Healthcare (1 mentions)
Imagequant tl, by GE Healthcare (1 mentions)
Mirvana mirna probe construction kit, by Thermo Fisher Scientific (1 mentions)
Halt protease inhibitor, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Santa Cruz Biotechnology (1 mentions)
β actin, by Abcam (1 mentions)
α 32p utp, by PerkinElmer (1 mentions)
48 protocols using storm 840 phosphorimager
1
Quantitative ATPase Activity Assay
2
ADP Binding Assay for hRAD51 with mHOP2-MND1
3
ChIP Assay and Quantification Protocol
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ChIP assay and its quantification in all figures except for Fig 2D were carried out as described previously [123 (link)–125 (link)] with modifications. Briefly, exponentially growing cells were fixed in 3% paraformaldehyde, and chromatin was sheared into 500 to 700-bp fragments using a Misonix Sonicator 3000 (Qsonica, Newtown, CT). Chromatin-associated proteins were then immunoprecipitated using mouse monoclonal anti-V5/Pk SV5-Pk1 (AbD Serotec, Kidlington, UK) or anti-FLAG M2 (Sigma-Aldrich, St. Louis, MO) antibodies in combination with Protein G-coupled Dynabeads (Life Technologies, Carlsbad, CA). DNA extracted from the immunoprecipitates was subjected to PCR analysis, and the PCR products were separated on a 4% polyacrylamide gel. The gel was stained with SYBR Green I (Life Technologies) and analyzed with Storm 840 Phosphorimager (GE Healthcare). Relative enrichment of the target sequences was calculated by multiplex PCR including primers that amplify a gene-free region (GFR) [126 (link)] as internal control as described previously [123 (link)–125 (link)]. ChIP assay described in Fig 2D was performed using the anti-myc 9E10 monoclonal antibodies (Cell Signaling Technology, Danvers, MA) as previously described [90 (link), 92 (link), 127 (link)]. PCR primers used in our ChIP studies are listed in S2 Table .
4
Rad52-Mediated Annealing Assay
5
Southern blot analysis of telomeric and LacO repeats
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Genomic DNA was digested overnight with the indicated restriction enzymes, and separated on an agarose gel using 1x Tris-Acetate-EDTA buffer. DNA from the agarose gel was transferred to a Hybond-XL membrane (GE Healthcare, Little Chalfont, UK) using buffer containing 0.5 M NaOH and 1.5 M NaCl. The membrane was then UV cross-linked using an XL-1000 UV Crosslinker (Spectronics, Westbury, NY) and incubated with a DNA probe labeled with [α-32P] dCTP. Hybridization was carried out overnight in Church buffer at 65°C as described [122 ]. Membranes were exposed for 2 days to a Phosphorimager screen, and detection of telomere or LacO repeats was done using a Storm 840 Phosphorimager (GE Healthcare). The DNA probe for the detection of telomere repeats by Southern blotting has been described previously [56 (link)]. For detecting LacO repeats, the 316-bp XbaI fragments that contain LacO repeats were excised from the pSV2-DHFR-8.32 plasmid [61 (link)] and used as a probe. The 448-bp PvuII fragment from the pJK148 backbone [116 (link)] was used as a probe to detect the DNA fragment containing the internal telomere tract.
6
Transcriptional Regulation of Ty1 Retrotransposons
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For strains carrying pBDG606, 10 ml of SC-Ura + 2% raffinose was inoculated with a single colony, and the culture was grown at 30° to an OD600 of 2, or up to 24 hr, depending on the strain; 2 ml of the raffinose culture was centrifuged and cells were suspended in 10 ml of SC-Ura + 2% galactose and grown at 22° for 16 hr. To detect endogenous Ty1i RNA, a single colony was suspended in 5 ml YEPD and grown at 30° overnight. The overnight culture was diluted different amounts depending on the growth of each strain (1:10 for WT, 1:4 for loc1 Δ, 1:5 for rps0b Δ, and 1:8 for rpl7a Δ) in a total of 10 ml fresh YEPD and grown at 22° for 8 hr. Total RNA was extracted using the MasterPure yeast RNA purification kit (Epicentre Biotechnologies, Madison, WI) with modifications as described previously (Saha et al. 2015 (link)). Poly(A)+ RNA was isolated from 250 μg total RNA using the NucleoTrap mRNA purification kit (Clontech, Mountain View, CA) following manufacturer’s protocol. Northern blot analysis using 32P-labeled riboprobes was performed as described previously (Saha et al. 2015 (link)). Hybridization signals were visualized and quantified using a STORM 840 phosphorimager and ImageQuant software (GE Healthcare).
7
ADP Binding Assay for hRAD51 with mHOP2-MND1
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The reaction mixtures contained 25 mM Tris acetate, pH 7.5, 0.2 mM ADP, 5 μCi [14C]ADP, 0.1 mM magnesium acetate, 2 mM DTT, BSA (100 μg/ml), and ssDNA (#90; 30 μM, nucleotides), 0.7 mM Al(NO3)3 or BeSO4, and 8 mM NaF.
First, mHOP2-MND1 (1 μM) was incubated with ssDNA for 5 min at 37 °C. Then, hRAD51 (10 μM) was added and incubation continued for 15 min. The resulting nucleoprotein complexes were subjected to vacuum filtration through Nylon membrane (Schleicher & Schuell). To remove unbound [14C]ADP, the membrane was washed twice with buffer containing 25 mM Tris acetate, pH 7.5, and 0.1 mM magnesium acetate. The membrane was dried and the amount of hRAD51-bound ATP was visualized and quantified using a Storm 840 PhosphorImager and ImageQuant 5.2 software (GE Healthcare).
First, mHOP2-MND1 (1 μM) was incubated with ssDNA for 5 min at 37 °C. Then, hRAD51 (10 μM) was added and incubation continued for 15 min. The resulting nucleoprotein complexes were subjected to vacuum filtration through Nylon membrane (Schleicher & Schuell). To remove unbound [14C]ADP, the membrane was washed twice with buffer containing 25 mM Tris acetate, pH 7.5, and 0.1 mM magnesium acetate. The membrane was dried and the amount of hRAD51-bound ATP was visualized and quantified using a Storm 840 PhosphorImager and ImageQuant 5.2 software (GE Healthcare).
8
Northern Blot Analysis of Viral and Host RNA
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Total RNA was isolated as described (6 (link)) at 14 or 18 h postinfection (hpi) as indicated. Templates for Northern probes were amplified by PCR from viral or mouse genomic DNA (using PCR primers listed in Table S2 in the supplemental material), prepared as described in reference 6 (link) for probes 2 and 7 (formerly EGR 26 probes 1 and 4 [6 (link)], respectively), or obtained from a commercial vendor for actin (Life Technologies). Northern blotting using Ambion’s NorthernMax kit (Life Technologies, Grand Island, NY) and generation of single-stranded P32-labeled RNA probes using the Maxiscript Sp6/T7 kit (Life Technologies) were performed as described previously (6 (link)). Probe 12 was generated using the mirVANA miRNA probe construction kit (Life Technologies) according to the manufacturer’s instructions. Five micrograms of total RNA was used for all Northern blot analyses unless otherwise stated. Membranes were scanned using a Storm 840 Phosphorimager and quantitated using ImageQuant TL (GE Healthcare Biosciences, Pittsburgh, PA). For quantitation of each sample, the indicated transcript signal was normalized to the signal from the actin loading control.
9
RAD51-Mediated D-Loop Formation Assay
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The nucleoprotein filaments were formed by incubating RAD51 protein with 32P-labeled ssDNA (#90, 90-mer) in buffer containing 35 mM Tris-HCl, pH 7.5, 2 mM ATP, 100 µg ml−1 BSA, 1 mM DTT, 20 mM KCl (from the protein stock), 1.5 mM MgCl2, 0.5 mM CaCl2, and the ATP-regenerating system (30 U ml−1 creatine phosphokinase and 20 mM creatine phosphate for 30 min at 37 °C. The reaction was transferred to 30 °C followed by addition of RAD54 and supercoiled pUC19 dsDNA (50 µM nucleotide or 9.3 nM molecules) to initiate D-loop formation. Aliquots (10 µl) were withdrawn at indicated time points, and D-loops were deproteinized by treatment with 5 µl of stop solution (1.36% SDS, 1.4 mg ml−1 proteinase K, 6% glycerol, 0.015% bromophenol blue) for 15 min at 37 °C; and analyzed by electrophoresis in 1% agarose-TAE (40 mM Tris-acetate, pH 8.0, and 1 mM EDTA) gels. The gels were dried on DE81 chromatography paper and quantified using a Storm 840 PhosphorImager (GE Healthcare).
10
Spiro Inhibits RAD51-Mediated DNA Strand Exchange
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The effect of Spiro on the RAD51 strand exchange activity was measured using the D-loop assay (28 ,29 ). Human RAD51 (0.3 μM) was incubated with Spiro (at indicated concentrations) in buffer containing 25 mM Tris-acetate (pH 7.5), 1 mM ATP, 1 mM CaCl2, 100 μg/ml BSA, 2 mM DTT, and 20 mM KCl (added with the protein stock) and 2% v/v DMSO (added with Spiro) for 10 min at 37°C. Then 32P-labeled 90-mer ssDNA (oligo#90) (0.9 μM, nt) was added and the nucleoprotein filaments were formed for 15 min at 37°C. D-loop formation was initiated by addition of pUC19 supercoiled dsDNA (15 μM, nt) and carried out for 15 min at 37°C. The DNA products were deproteinized by treatment with 1 mg/ml proteinase K in stop mixture containing 1% SDS, 6% glycerol and 0.01% bromophenol blue for 15 min at 37°C, and analyzed by electrophoresis in a 1% agarose gel in 1XTAE buffer (40 mM Tris acetate, pH 8.3 and 1 mM EDTA) at 5 V/cm for 1.5 h. The gels were dried on DEAE-81 paper (Whatman) and quantified using a Storm 840 PhosphorImager and ImageQuant 5.2 (GE Healthcare) (28 ,29 ).
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