SeqA-DNA複合体の結晶化のためのDNA二重鎖の反復最適化

This experiment details how to obtain diffraction quality crystals of a protein bound to DNA containing a tandem hem methylated.GATC. Repeat to favor crystal packing of the protein DNA complex. Start with the rational design of the DNA length, the GATC spacing and the DNA ends proceed with the complementary single stranded oligonucleotides to purify the DNA and then anele to form the hemi methylated DNA duplex, then mix in the purified seq A to form the complex.

Next, find optimal conditions to grow diffraction quality crystals of the protein DNA complex with crystallization trials using commercial sparse matrix screens. Demonstrated results. Share the effect of varying DNA length GATC spacing and ends on the diffraction quality of the CQ A DNA crystals.

The goal of this work is to obtain diffraction quality crystals of CK bound to DNA, but it can also help to understand the general variables that need to be considered when designing DNA duplexes for crystallization of other protein DNA complexes demonstrating this procedure will be used in Chang, A for doctoral fellow from my lab. This study uses a variant of the DNA replication regulatory proteins. Seek a that forms stable dimers and has a shortened linker region between its ization and DNA binding domains.

This variant restricts DNA binding to tandem GATC repeats separated exclusively by one turn on the DNA first transform BBL 20 1D three cells with the plasmid encoding DEMETRIC seq A under the control of the T seven promoter plate. The transformation reaction on LB agar plates containing 100 micrograms per milliliter, ampicillin and place in the bacterial incubator overnight. Pick mixed colonies and start a small scale overnight culture.

The next morning inoculator one liter culture with 10 milliliters of the overnight inoculum. When the culture is in log phase, add two milliliters of 0.5 molar IPTG to induce protein production. Continue the incubation for three hours in a 37 degree Celsius orbital shaker.

Then harvest the cells by centrifugation at 3, 300 G for 10 minutes. Reese, suspend the cell palette in purification, buffer a lies by sonication and clear the lysate by centrifugation at 39, 000 times G for 40 minutes. Now load the S supernatant onto a heparin column equilibrated with purification buffer.

To elute the CK protein. Use a linear gradient to one molar sodium chloride CK protein elutes at around 0.7 molar sodium chloride. Pull the CK containing fractions and dilute to lower the ion strength of the sample.

Then load the sample into a cation exchange chromatography column E equated with purification buffer. Apply a linear salt gradient to loop pure CK protein at around 0.4 molar sodium chloride. Combine the CK containing fractions.

Concentrate to three milligrams per milliliter and store in storage. Buffer design complimentary unmethylated and methylated oligonucleotides. Prepare samples of one micromole of each lyophilized single strand DNA in 800 microliters of autoclave, double distilled water vortex and set aside for 15 minutes now and 800 microliters.

Have preheated two times loading buffer to each sample for 20 to 30 nucleotides long oligonucleotides prepare a large 10%denaturing gel. Once polymerized, remove the comb and rinse the well thoroughly. Assemble the gel filling the top and bottom reservoirs with one times TBE running buffer.

Pre-run the gel at 750 volts to warm up the gel to 55 degrees Celsius. Then stop the run and rinse the wells thoroughly with running buffer. Now heat the oligonucleotides to 90 degrees Celsius for two minutes.

Vortex and spin. The samples proceed immediately to load the gel. Run the gel at around 700 volts until the oligonucleotide has migrated.

Halfway On a 10%poly acrylamide gel alph phenol blue co migrates with oligonucleotides around 20 bases long and xylene syl ff with around 60 bases long. Disassemble the gel from the apparatus and remove the spacers on a flat surface. Remove one glass plate and cover the gel with plastic wrap.

Then flip the gel, remove the other glass plate and cover the gel with plastic wrap. Next, mark the bands using UV light and a fluorescent plate behind the gel. To see the DNA shadow with a razor blade, excise the band and cut it into small pieces.

Transfer each sample into a sterile 15 milliliter tube. Add nine milliliters of elucian buffer and dilute overnight at 37 degrees Celsius with agitation. Carefully transfer the solution to an autoclave centrifuge tube using a gel loading pipette tip.

To avoid transferring acrylamide pieces, add one milliliter of three milli sodium acetate a pH seven plus 25 milliliters of chilled 100%ethanol incubate at minus 20 degrees Celsius for at least three hours. Centrifuge the precipitated DNA. Then dry the pellets on the speed vac at medium heat resuspend each pellet in 400 microliters of autoclave, double distilled water and transfer to a fresh tube.

Add 40 microliters of three molar sodium acetate at pH seven and one milliliter of 100%ethanol vortex and incubate for 30 minutes at minus 20 degrees Celsius after centrifugation for 15 minutes at 18, 000 times. G, rinse the DNA pellets with 100 microliters of 70%cold ethanol to remove any residual salt spin for six minutes at 18, 000 times. G then discard the ethanol and dry the pellet on a speed vac.

Now resend each pellet in 100 microliters of autoclave double distilled water. Determine the concentration of the oligonucleotides by spectrometry. To prepare the hemi methylated DNA duplexes first mix equal molo concentrations of the complementary single strands.

Then heat the mixtures to 95 degrees Celsius in a water bath for five minutes To anil the strands. Let the samples cool down slowly to room temperature Inside the water bath. Combine equal volumes of 81 micromolar purified CQ a protein and 81 micromolar hem methylated DNA.

Then incubated room temperature for 15 minutes. Store at four degrees Celsius until use screen for crystallization conditions using commercial sparse matrix screens. Once initial crystallization leads have been identified, optimize the conditions to grow diffraction quality crystals To cry and protect the resulting CK DNA crystals.

Either increase the amount of PEG 400 present in the crystallization solution to a final concentration of 25%or add 20%glycerol to the crystallization solution. Scoop out individual crystals with nylon loop flash. Freeze the samples in liquid nitrogen.

Now test the diffraction limit of each crystal at 100 k. To obtain the crystal structure of the shortened mutant of CK bound to Hemi methylated DNA, we consecutively optimize three parameters on the DNA, the spacing between Hemi methylated GATC sequences, the length of the duplex and the absence or presence of five prime overhangs. This study use a DME variant of seek a with a point mutation in the end terminal domain that prevents further a ligamentization and a shortened linker that restricts DNA binding to tandem.

GATC repeats separated exclusively by one turn on the DNA electromobility shift assays indicate that the modified CK protein preferentially binds GATC repeats separated by nine to 10 base pairs. Therefore, initial screens utilize duplexes 23 to 24 base pairs long containing two hem methylated. GATC repeats separated by either nine or 10 base pairs.

Three duplexes yielded nicely shaped crystals. While none of the crystals defracted to high resolution. The data did indicate that A-G-A-T-C separation of nine base pairs was preferred for crystallization unexpectedly enhancing the groove backbone interactions by including a CG dinucleotide between the two GATC sites did not alter the diffraction limit of the crystals.

By contrast, optimizing the length of the overhangs dramatically changed the molecular contacts and the crystal morphology. The crystal structure of this CK protein bound to a hem methylated DNA duplex confirmed that the free face of the DNA duplex does not engage in crystal contacts despite the relative size of protein and DNA, most interactions between symmetry mates are mediated by protein, protein and protein DNA interactions. Interestingly, in this particular case, the beneficial effect of a five prime dinucleotide overhang is not due to the formation of a pseudo continuous DNA.

Instead, the five prime end of the methylated strand projects away from the DNA axes and interacts with the proximal CK molecule of the complex, explaining why two nucleotides were strictly required to change the crystal packing. After watching this video, you should have a good understanding of how to purify proteins and design DNA duplexes for protein DNA complex crystallization. It's important to remember that although this protocol was device to crystallize the CK DNA complex, the rational optimization of DNA length sequence and ends provides a general framework for the design of DNA duplexes for the crystallization of other protein DNA complexes.