Studying Ribonucleotide Incorporation: Strand-specific Detection of Ribonucleotides in the Yeast Genome and Measuring Ribonucleotide-induced Mutagenesis

This method can help answer questions such as how many ribonucleotides are incorporated into nascent, leading, and lagging strand DNA in the genome. Though this method can provide quantitative and qualitative information regarding ribonucleaotide density in the yeast genome it can also be applied to other systems such as mammalian genomes. The alkaline gel electrophoresis provides a semi quantitative view of ribonucleotide incorporation in the whole genome and strand specific southern blotting allows for greater sensitivity and quantitation at a specific genomic locus.

To begin this procedure, add six microliters of one molar potassium hydroxide to each sample of freshly prepared yeast genomic DNA and incubate at 55 degrees celsius for two hours. After two hours, incubate the samples on ice for five minutes. Then add to each sample four microliters of 6-6 alkaline DNA loading buffer.

Cast an alkaline gel comprised of one percent agarose, 50 millimolar sodium hydroxide and one millimolar EDTA pH eight with a comb that allows for 24 microliters of DNA sample to be loaded per lane. Spin down the tubes of samples briefly. Load the entire 24 microliters of each sample into the alkaline agarose gel.

Include an appropriate DNA size marker. Electrophorese at 30 volts for 30 minutes. Then lower the voltage to 10 volts and electrophorese for 18 to 20 hours.

On the following day, neutralize the gel. Immerse the gel in neutralization buffer one and incubate at room temperature with gentle agitation for 45 minutes. Replace the neutralization buffer blend with fresh buffer.

And incubate for another 45 minutes. To stain the DNA, soak the neutralized gel in 200 milliliters of water containing a one to 10000 dilution of sybr gold stain at room temperature with gentle shaking for two hours. Visualize the DNA using a UV transilluminator.

The DNA from the alkaline gel will be transferred to a positively charged nylon membrane by capillary action. First soak the alkaline gel at room temperature for 15 minutes in alkaline transfer buffer. Next, wet the nylon membrane briefly with water and soak it in alkaline transfer buffer for five minutes.

Set up the transfer platform by inverting three 100 milliliter glass beakers in a glass baking dish that is slightly larger than the size of the agarose gel. Set a glass plate on top of the glass beakers. Drape two large pieces of three MMCHR chromatography paper over the glass plate.

Pour alkaline transfer buffer over the chromatography paper and half fill the glass dish. Smooth out the bubbles using a five milliliter glass pipette. Place the agarose gel on top.

Again, smoothing out the bubbles. Surround all edges of the gel with parafilm. Pour a small amount of alkaline transfer buffer onto the gel.

Place the presoaked nylon membrane on top of the gel and smooth out the bubbles. Put two pieces of chromatography paper cut to the size of the agarose gel. Place them on top of the membrane and smooth out any bubbles.

Place a large stack of paper towels on top of the transfer sandwich and carefully place a glass plate on top of the paper towels. Add a weighted object to the top and let the transfer proceed at room temperature overnight. On the following day, carefully take the transfer stack apart.

Soak the membrane at room temperature in neutralization buffer two for 15 minutes. Cross link the DNA to the charged nylon membrane using a UV cross linker. Use the auto cross link setting.

Prior to starting the hybridization procedure, prepare the radiolabeled probe as described in the text protocol. Roll the wetted membrane into a hybridization tube and add 25 milliliters of freshly prepared hybridization buffer. Incubate a 65 degrees celsius with rotation in a hybridization oven for one to two hours.

Carefully pour off the solution and add 25 milliliters of hybridization buffer plus the probe. Incubate at 65 degrees celsius for 16 to 18 hours with rotation. Next, pour off the solution into a radioactive waste container.

Add phosphate STS washing solution one to the hybridization tube and rotate at room temperature for five minutes. In this manner, perform five washes of five minutes each with phosphate STS washing solution one. After the fifth wash with phosphate STS washing solution one wash the membrane twice with phosphate STS washing solution two for 15 minutes each time at 65 degrees celsius with rotation.

Use tweezers to remove the membrane from the hybridization tube and place the membrane in an opened plastic sleeve protector. Cover and expose to an imaging plate. If necessary, strip and reprobe the blot with a different probe.

To strip, cover the membrane with 25 milliliters of a 50 percent formamide 2XSSPE solution and incubate at 65 degrees celsius for two hours. Pour off the solution into a radioactive waste container. Add phosphate STS washing solution two to the hybridization tube, and rotate at 65 degrees celsius for 15 minutes.

Check the membrane with a geiger counter to confirm the absence of signal before performing the prehybridization and hybridization as demonstrated earlier. These gel images have yeast genomic DNA treated with or without potassium hydroxide show that the cells lacking RNH2 retain more ribonucleotides in the genome. The M644L variant of polymerase two reduces the ability to incorporate ribonucleotides while the M644G mutant incorporates more ribonucleotides than the wild type polymerase.

An alkaline agarose gel can be further probed by strand specific southern blot analysis. In this example, the probes anneal to the nascent leading DNA strand within the URA3 reporter gene that has been inserted close to ARS306 in two opposite orientations. Representative results of southern blotting using the nascent leading strand specific probes show a DNA fragmentation pattern caused by alkali cleavage at ribonucleotides and nascent leading strands DNA in a wild type strain and in strains expressing the variants of polymerases alpha, delta, and epsilon that are promiscuous for ribonucleotide incorporation.

This graph shows the quantification of the radioactive signal by fraction of the total in each lane from the southern blot. While attempting the procedure, it is important to remember to always use freshly isolated DNA from lock face cells. After its development, this technique paved the way for researchers in the field of DNA repair to quantify the density and the strand into which ribonucleotides have been incorporated by the replicative DNA polymerases.

Following this procedure, other methods like hydrolated end sequencing or HydEn-seq, can be performed in order to answer additional questions like the location of the ribonucleotides in the genome. Don't forget that working with radioactive materials can be extremely hazardous and precautions should always be taken to avoid contamination of the workbench and equipment while performing this procedure.