Single molecule methods have been used to study telomeric protein DNA interaction. However, preparing single molecule constructs with the telomeric repetitive motif remains a challenging task. In this protocol, we're outlining the steps for expressing and then purifying TRF2 protein, preparing telomeric DNA, setting up single molecule mechanical assays, and analyzing the resulting data.
Single molecule tools are powerful techniques for exploring telomeric protein DNA interactions. Single molecule mechanical methods such as magnetic tweezers, optical tweezers, and the AFM have been used to investigate TRF2-dependent DNA distortion, reveal TRF2 media to columnar stocking of human telomeric chromatin, and observe presence of telomerase catalysis, among other applications. We investigated telomeric DNA protein interactions using single molecular magnetic tweezers, enabling precise measurements of changes in extension under the durations of protein DNA interactions under applied forces.
From these measurements, we derived the dissociation kinetics of the telomeric DNA protein complexes. Furthermore, the formation of loops within these complexes was revealed. Numerous structures of telomeric binding proteins in complex with DNA have been resolved using Cryo EM, X-Ray Crystallography, and NMR.
These structural methods have advanced our understanding of telomeric protein DNA interactions. To further investigate the dynamics of these interactions, we have developed a single molecule mechanical method specifically for starting telomeric protein DNA interactions. Single molecule tools are powerful techniques for investigating protein DNA interactions at telomerase, with methods especially viable for protein topological confirmations and analyzing the kinetics of protein DNA association and dissociation.
To begin the transformation of Escherichia coli BL21 DE3 cells, thaw 50 microliter aliquot of competent cell suspension on ice. Add one microliter of DNA of pET28a-SUMO TRF2 plasmid to the competent cell suspension, and gently swirl the tube to mix the suspension. After transformation, spread 100 microliters of the cell suspension on a Luria-Bertani or LB auger plate containing 50 micrograms per milliliter kanamycin.
Incubate the cells overnight at 37 degrees Celsius. The next day, pick a colony of the transformed cells and culture it in five milliliters of LB medium supplemented with 50 micrograms per milliliter kanamycin. Incubate for 18 hours at 37 degrees Celsius with shaking at 220 RPM.
After incubation, induce the culture with one millimolar IPTG as a final concentration. Incubate at 20 degrees Celsius for 17 hours to promote protein expression. Load the crude protein extracts along with the loading buffer onto an SDS-PAGE gel.
Run the samples initially at 100 volts for 30 minutes, followed by 120 volts for 50 minutes in a Tris-Glycine buffer. Then, stain with Coomassie blue to visualize protein expression. For protein purification, centrifuge the crude protein extract at 38, 000G for 40 minutes at four degrees Celsius, and filter the supernatant through a 0.22 micrometer syringe filter.
Load the filtered supernatant onto the pre-equilibrated nickel column in the binding buffer. Wash the column with binding buffer and dilute the protein with the prepared imidazole gradient, collecting 12 fractions of one milliliter each. Add glycerol to a final concentration of 50%to the concentrated and buffer-exchanged protein for storage.
The expression of TRF2 in Escherichia coli was successfully demonstrated as shown by SDS-PAGE, where distinct bands corresponding to TRF2 appeared before and after induction. To begin, centrifuge one times 10 to the power of seven HeLa cell lines containing telomeric restriction fragments DNA at 1, 000G for three minutes. Discard the supernatant and resuspend the pellet in 200 microliters of PBS.
After SDS proteinase K and sodium chloride treatment, centrifuge the cell suspension at 16, 900G for 10 minutes. Transfer the supernatant to a new centrifuge tube, and add an equal volume of isopropanol, avoiding floating lipids or sediment. Centrifuge at high speed and wash the pellet with 500 microliters of 70%ethanol.
After drying the DNA pellet, carefully resuspend it in 475 microliters of TE buffer and gently mix by tapping the bottom of the tube. Now, add 25 microliters of 10 milligrams per milliliter RNase A, and tap the tube until the pellet is completely dissolved. Next, add 1/10 volume of three molar sodium acetate and two volumes of cold 100%ethanol, and place the tube at minus 80 degrees Celsius for two to three hours.
After centrifugation and 70%ethanol washes, add 100 microliters of TE buffer to the DNA pellet. Tap the tube to mix, and place it at four degrees Celsius for two hours to fully dissolve. For DNA digestion, combine four micrograms of genomic DNA with one microliter of CVIA 2, two microliters of 10X digestion buffer, and water to reach a total volume of 20 microliters.
Incubate the mixture at 25 degrees Celsius for 12 hours. In a thermal cycler, heat the mixture at 75 degrees Celsius for three minutes, then, decrease the temperature by 0.1 degrees Celsius every 30 seconds until reaching 25 degrees Celsius. After cleaning and drying, coat the bottom cover slips with 20 microliters of 0.1%nitrocellulose, and add reference beads.
Bake the cover slips at 100 degrees Celsius for four minutes. Assemble the flow cell sandwich, and after heating it 85 degrees Celsius, use two swabs to massage the assembly until the parafilm seals the channel. Wash 10 microliters of M270 beads five times with 50 microliters of working buffer using a magnet.
After washing, add the beads on top of the digested genomic DNA into a 1.5 milliliter centrifuge tube. Gently flick the tube a few times to mix the beads and DNA sample. Leave the mixture on ice for one hour.
After incubation, wash the mixture with 500 microliters of working buffer three times using a magnet to pull the beads down, with 10 minute intervals between washes. Resuspend the sample in a working buffer, and load 30 microliters of the mixture into the flow cell. Flush the unbound magnetic beads after 30 minutes.
After placing the flow cell on top of the objective lens, select a pair of five millimeter cubic magnets arranged in a vertical configuration and align the magnet holder with the X axis of the magnetic tweezer's light path for imaging. Launch the graphical programming software, and connect the controllers for the magnetic tweezers. Adjust the field of view to locate a reference bead at the bottom of the flow cell, and adjust the objective lens slightly so that the reference bead shows clear diffraction rings.
Write a script in MATLAB to control motor movements for force ramp assays. Import the script into graphical programming software to test the single molecule experiments. At a slow flow rate, load 200 microliters of 10 nanomolar TRF1 into the flow cell.
After 30 minutes of binding, choose a script for a force ramp experiment with a force loading rate of plus minus one piconewton per second. Name the data files, and run the experiment. Genomic DNA integrity was confirmed using agarose gel electrophoresis, with the resulting TRFs displaying consistent lengths across various human cell lines.
Telomeric repeat sequences in TRFs were detected via southern blotting, showing clear hybridization signals. Force extension curves from the force ramp assay showed zigzag patterns during stretching, indicating the breaking of protein DNA interactions. The dissociation kinetics of telomeric DNA protein complexes showed a linear relationship between force and dissociation rate.
Moreover, the length heterogeneity of telomeric DNA from human cells investigates the loop formation mechanism in telomeres of various lengths.
