32p atp

Reactions (10 μl) contained 40nM of Leishmania infantum RAD50 or RAD50^K42A (purified by double affinity purification accordingly to [99 (link)]) in 50mM Tris-HCl pH 7.5, 1mM Mg(CH₃COO)₂, 1mM DTT and 100 μg/ml BSA supplemented with 50 nCi [^ɣ-32P]ATP (3000 Ci/mmole; Perkin Elmer Life Sciences). Aliquots (5 μl) were removed, stopped by addition of EDTA, and the percentage of ATP hydrolyzed was determined by thin layer chromatography followed by quantification using a Fujifilm Phosphoimager.

DNA fragments were end-labeled with ³²P using T4 polynucleotide kinase and [ɣ-³²P]ATP. As our objective was to build a four-stranded structure, it was found important not to use ATP of high specific activity. The ideal would be to have a single ³²P atom in each structure built, as the decay of a ³²P atom into ³²S quickly result in a nick due to the instability of the sulfodiester bond [28 (link)]. If a structure contains several ³²P atoms, the decay of any one of them leads to the formation of a labeled contaminant, whereas when the four-stranded structure contains a single ³²P atom the decay product will not be radioactive and will not be visible upon autoradiography (note that one should remain aware of the fact that it is present, a phenomenon that is unavoidable when using radioactive isotopes).
For this reason, [ɣ-³²P]ATP (Perkin-Elmer, 3000 Ci/mmol, i.e. containing ~ ⅓ ³²P and ~ ⅔ ³¹P at position ɣ) was diluted with non-radioactive ATP in a proportion such that the mixture contained 10% radioactive ATP only. Thus, the probability that a four-stranded structure would contain two ³²P atoms was low, and decay contaminants were hardly visible.
After labeling, the DNA fragments were extracted with chloroform-isoamyl alcohol as described above, precipitated twice with ethanol to get rid of the unincorporated radioactivity, and redissolved in TE buffer.

EMSA was performed as previously described (35 (link)). Briefly, nuclear protein extracts were prepared using a nuclear extract kit (Active Motif, Carlsbad, CA), following the manufacturer’s protocol. Probe labeling was accomplished by treatment with T4 kinase (Life Technologies, Burlington, ON, Canada) in the presence of [³²P]ATP (Perkin Elmer, Waltham, MA). Labeled oligonucleotides were purified on a Sephadex G-25M column (GE Healthcare, Pittsburgh, PA). Nuclear protein (10 μg) was added to a 10-μl volume of binding buffer supplemented with 1 μg poly(dI:dC) (GE Healthcare) for 15 min at room temperature. Labeled double-stranded oligonucleotide was added to each reaction mixture, incubated at room temperature for 30 min, and separated by electrophoresis on a 6% polyacrylamide gel in 0.5× Tris-boric acid-EDTA buffer. Gels were vacuum-dried and subjected to autoradiography. The following synthesized double-stranded oligonucleotides were used: NF-κB consensus sequence on the IL-6 promoter (5′-AGTTGAGGGGACTTTCCCAGGC-3′) (Promega, Madison, WI) and NFAT-binding consensus sequence on the mouse IL-13 promoter (5′-AAGGTGTTTCCCCAAGCCTTTCCC-3′) (Sigma-Aldrich).

ProF (3,6-Diaminoacridine hydrochloride) was purchased from Sigma-Aldrich (MO, USA). TO was purchased from Sigma-Aldrich. C-di-GMP and c-di-AMP were synthesized following literature and obtained as triethylammonium acetate salt [48 (link)]. ³²P-GTP or ³²P-ATP was purchased from Perkin Elmer (MA, USA). Images were obtained by STORM scanner and quantified by ImageQuant software (Molecular Dynamics). Fluorescence was measured by Cary Eclipse Fluorescence Spectrophotometer. AFM image was taken by Veeco Multimode AFM with nanoscope III controller with tapping mode. The visualization probe was Silicon AFM Probes TAP 300 from Ted Pella, Inc. Fluorescence lifetime was measured using time-domain system integrated with fluorescence lifetime imaging microscope (FLIM) system Alba V (ISS, IL, USA). The system is equipped with SPC-830 TCSPC module and pulsed laser system (Becker and Hickl GmbH). Laser BHL-473 nm and observation through band pass filter 514/50 nm was used for TO. Data analysis was performed using Vista Vision software v. 218 from ISS.