We thank all current and former Kunkel Lab members for their work and discussions related to protocol and reused data presented here. This work was supported by Project Z01 ES065070 to T.A.K. from the Division of Intramural Research of the National Institutes of Health (NIH), National Institute of Environmental Health Sciences (NIEHS).

1. Alkaline Hydrolysis and Strand-specific Southern Blot (Figure 1)
<ol>
	<li>Cell growth and genomic DNA isolation
	<ol>
		<li>Isolate yeast genomic DNA using a yeast DNA purification kit following the manufacturer&#8217;s instructions. Use the cultures that are in the exponential phase of growth (between an optical density of 0.5 and 1). Resuspend the final DNA pellet in 35 mL of 1x TE buffer (10 mM Tris-HCl, pH 7.5, 1 mM ethylenediaminetetraacetic acid (EDTA)).</li>
		<li>Add 1 mL of 5 ...

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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topoisomerase+1-mediated+removal+of+ribonucleotides+from+nascent+leading-strand+DNA.">Topoisomerase 1-mediated removal of ribonucleotides from nascent leading-strand DNA.</a> Molecular Cell. 49 (5), 1010-1015 (2013).</li><li>Clausen, A. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+replication+enzymology+in+vivo+by+genome-wide+mapping+of+ribonucleotide+incorporation.">Tracking replication enzymology in vivo by genome-wide mapping of ribonucleotide incorporation.</a> Nature Structural &amp; Molecular Biology. 22 (3), 185-191 (2015).</li><li>Foster, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+determining+spontaneous+mutation+rates.">Methods for determining spontaneous mutation rates.</a> Methods Enzymology. 409, 195-213 (2006).</li><li>Williams, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+that+processing+of+ribonucleotides+in+DNA+by+topoisomerase+1+is+leading-strand+specific.">Evidence that processing of ribonucleotides in DNA by topoisomerase 1 is leading-strand specific.</a> Nature Structural &amp; Molecular Biology. 22 (4), 291-297 (2015).</li><li>Pavlov, Y. I., Shcherbakova, P. V., Kunkel, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+consequences+of+putative+active+site+mutations+in+yeast+DNA+polymerases+alpha,+epsilon,+delta,+and+zeta.">In vivo consequences of putative active site mutations in yeast DNA polymerases alpha, epsilon, delta, and zeta.</a> Genetics. 159 (1), 47-64 (2001).</li><li>Nick McElhinny, S. A., Kissling, G. E., Kunkel, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+correction+of+lagging-strand+replication+errors+made+by+DNA+polymerases+{alpha}+and+{delta}.">Differential correction of lagging-strand replication errors made by DNA polymerases {alpha} and {delta}.</a> Proceedings of the National Acaddemy of Sciences U S A. 107 (49), 21070-21075 (2010).</li><li>Clark, A. B., Lujan, S. A., Kissling, G. E., Kunkel, T. 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Here, we describe the protocols for two sets of experiments that are frequently used to semi-quantitatively analyze ribonucleotides incorporated during DNA replication and the mutagenic effects of unrepaired ribonucleotides. Although these approaches involve the model eukaryote S. cerevisiae, these techniques can be easily adapted to other microbes and even higher eukaryotes.
Probing for unrepaired ribonucleotides in DNA using alkaline-agarose electrophoresis coupled with Southern blo...

During eukaryotic nuclear DNA replication, ribonucleotides are incorporated into the genome by all three major DNA replicases, DNA polymerases (Pols) &#945;, &#949;, and &#948;1,2. RNase H2-dependent ribonucleotide excision repair (RER3) removes the majority of these embedded ribonucleotides.
A ribonucleotide in DNA is susceptible to hydrolysis, as the 2&#39; hydroxyl group of the sugar moiety can attack the adjacent phosphodiester bond, releasing one end with a 2&#39;-3&#39; cyclic phosphate and the other with a 5&#39;-OH4. Alkaline conditions can greatly accelerate this reaction. Thus, the hydrolysis of embedded ribonucleotides during incubation in a basic solution causes fragmentation of genomic DNA, which can be visualized by alkaline-agarose electrophoresis5. This DNA can be transferred to a membrane and probed by Southern blot analysis using strand-specific probes that allow the identification of alkali-sensitive sites caused by ribonucleotides incorporated into the nascent leading- or lagging-strand DNA, respectively.
In yeast cells lacking RNase H2 activity, removal of ribonucleotides can be initiated when topoisomerase I (Top1) nicks the DNA at the embedded ribonucleotide6,7. However, when Top1 cleaves on the 3&#39; side of the ribonucleotide, this generates 5&#39;-OH and 2&#39;-3&#39; cyclic phosphate DNA ends that are refractory to religation. Failure to repair, or aberrant processing of these &#39;dirty ends&#39; can lead to genomic instability. In addition, if the incision occurs within a repeat DNA sequence, the repair process can lead to deletion mutations. This is particularly problematic for tandem repeats, where short deletions (of between two and five base pairs) are commonly observed in RNase H2-deficient cells. The Top1-dependent deleterious effects in the absence of yeast RNase H2 activity are exacerbated in a DNA polymerase&#160;&#949; mutant (pol2-M644G) promiscuous for ribonucleotide incorporation during nascent leading strand synthesis.
Processing of ribonucleotides in DNA leads to spontaneous mutations and this mutagenesis can be detected by using appropriate reporter genes and selecting for the accompanying phenotypic change. A fluctuation test or Luria and Delbr&#252;ck experiment is one of the most commonly used methods to measure spontaneous mutation rates using selectable reporter genes8,9. In yeast, the URA3 and CAN1 genes can be used as reporters in a forward mutation assay, which allows for the detection of all mutation types that result in the loss of gene function. The spontaneous mutation rate is estimated as the median of that observed for multiple parallel cultures started from single colonies without mutations in the target reporter gene. A yeast RNase H2-deficient strain such as rnh201&#916; has a moderately elevated overall spontaneous mutation rate that is largely caused by an elevated incidence of 2 - 5 bp deletions in tandem repeat sequences. Thus, to fully characterize the mutagenic effects of ribonucleotides in the genome, specific mutation rates need to be determined. In this case, the URA3 or CAN1 reporter genes can be amplified and sequenced to determine the types and locations of the mutations, and specific mutation rates can be calculated. Compiling mutations identified in multiple independent URA3 or CAN1 mutants can then be used to generate a mutation spectrum.

<table>
	<tbody>
		<tr>
			<td>YPD media</td>
			<td></td>
			<td></td>
			<td>20 g dextrose, 20 g peptone, 10g yeast extract, in deionized H2O up to 1 L, add 20 g Bacto agar for solid media, autoclave.</td>
		</tr>
		<tr>
			<td>COM plates</td>
			<td></td>
			<td></td>
			<td>1.3 g SC dropout mix, 1.7 g yeast nitrogen base without amino acids or (NH4)2SO4, 5 g (NH4)2SO4, 20 g dextrose, 20 g Bacto agar deionized H2O up to 1 L. Adjust pH to 5.8. Autoclave and 30 &#8211; 35 ml per plate.</td>
		</tr>
		<tr>
			<td>CAN plates</td>
			<td></td>
			<td></td>
			<td>1.3 g SC-Ura dropout powder, 1.7 g yeast nitrogen base without amino acids or (NH4)2SO4, 5 g (NH4)2SO4, 20 g dextrose, 20 g Bacto agar, 25 mg uracil and H2O up to 1 L. Autoclave for 15 mins at 121 &#176;C and cool down to 56 &#176;C. Add 6 mL of filter-sterilized 1% canavanine sulfate solution.</td>
		</tr>
		<tr>
			<td>5-FOA plates</td>
			<td></td>
			<td></td>
			<td>1.3 g SC-Ura dropout powder, 1.7 g yeast nitrogen base without amino acids or (NH4)2SO4, 5 g (NH4)2SO4, 20 g dextrose, 20 g Bacto agar, 25 mg uracil and H2O up to 800 mL. Autoclave for 15 mins at 121 &#176;C and cool down to 60 &#176;C. Add 200 mL of filter-sterilized 0.5% 5-FOA solution.</td>
		</tr>
		<tr>
			<td>L-Canavanine sulfate</td>
			<td>US Biological</td>
			<td>C1050</td>
			<td></td>
		</tr>
		<tr>
			<td>5-FOA</td>
			<td>US Biological</td>
			<td>F5050</td>
			<td></td>
		</tr>
		<tr>
			<td>20 mL glass culture tube</td>
			<td></td>
			<td></td>
			<td>Any brand</td>
		</tr>
		<tr>
			<td>Culture rotator in 30 &#176;C incubator</td>
			<td></td>
			<td></td>
			<td>Shaker incubator can be used instead</td>
		</tr>
		<tr>
			<td>96 well round bottom plates</td>
			<td></td>
			<td></td>
			<td>Sterile, any brand</td>
		</tr>
		<tr>
			<td>3 mm glass beads</td>
			<td>Fisher Scientific</td>
			<td>11-312A</td>
			<td>Autoclave before use</td>
		</tr>
		<tr>
			<td>12-channel pipettes</td>
			<td></td>
			<td></td>
			<td>Any brand</td>
		</tr>
		<tr>
			<td>Ex Taq DNA Polymerase</td>
			<td>TaKaRa</td>
			<td>RR001</td>
			<td></td>
		</tr>
		<tr>
			<td>Epicentre MasterPure Yeast DNA Purification Kit</td>
			<td>Epicenter</td>
			<td>MPY80200</td>
			<td></td>
		</tr>
		<tr>
			<td>3 M sodium acetate</td>
			<td>Sigma-Aldrich</td>
			<td>S7899</td>
			<td></td>
		</tr>
		<tr>
			<td>100% ethanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit 2.0 Fluorometer</td>
			<td>Invitrogen</td>
			<td>Q32866</td>
			<td>Newer model available</td>
		</tr>
		<tr>
			<td>dsDNA BR Assay kit</td>
			<td>Invitrogen</td>
			<td>Q32850</td>
			<td></td>
		</tr>
		<tr>
			<td>KOH</td>
			<td>Sigma-Aldrich</td>
			<td>221473</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>E7889</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll 400</td>
			<td>Dot Scientific Inc.</td>
			<td>DSF10400</td>
			<td></td>
		</tr>
		<tr>
			<td>Bromocresol green</td>
			<td>Eastman</td>
			<td>6356</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene cyanol FF</td>
			<td>International Technologies Inc.</td>
			<td>72120</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Sigma-Aldrich</td>
			<td>S8045</td>
			<td></td>
		</tr>
		<tr>
			<td>1 M Tris-HCl (pH 8.0)</td>
			<td>Teknova</td>
			<td>T5080</td>
			<td></td>
		</tr>
		<tr>
			<td>SYBR Gold Nucleic Acid Gel Stain</td>
			<td>Invitrogen</td>
			<td>S11494</td>
			<td></td>
		</tr>
		<tr>
			<td>UV transilluminator</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Amersham Nylon membrane Hybond-N+</td>
			<td>GE Healthcare</td>
			<td>RPN303B</td>
			<td></td>
		</tr>
		<tr>
			<td>3 MM CHR Chromotography paper</td>
			<td>Whatman</td>
			<td>3030-392</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Caledon</td>
			<td>7560-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Stratalinker 1800</td>
			<td>Stratagene</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick PCR Purification Kit</td>
			<td>Qiagen</td>
			<td>28106</td>
			<td></td>
		</tr>
		<tr>
			<td>G-25 spin column</td>
			<td>GE Healthcare</td>
			<td>27-5325-01</td>
			<td></td>
		</tr>
		<tr>
			<td>1 M Sodium phosphate buffer (pH 7.2)</td>
			<td>Sigma-Aldrich</td>
			<td>NaH2PO4 (S9638); 
			Na2HPO4 (S9390)</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Sigma-Aldrich</td>
			<td>L4522</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma-Aldrich</td>
			<td>A3059</td>
			<td></td>
		</tr>
		<tr>
			<td>Formamide</td>
			<td>Sigma-Aldrich</td>
			<td>47671</td>
			<td></td>
		</tr>
		<tr>
			<td>Geiger counter</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

studying ribonucleotide incorporation: strand-specific detection of ribonucleotides in the yeast genome and measuring ribonucleotide-induced mutagenesis

The presence of ribonucleotides in nuclear DNA has been shown to be a source of genomic instability. The extent of ribonucleotide incorporation can be assessed by alkaline hydrolysis and gel electrophoresis as RNA is highly susceptible to hydrolysis in alkaline conditions. This, in combination with Southern blot analysis can be used to determine the location and strand into which the ribonucleotides have been incorporated. However, this procedure is only semi-quantitative and may not be sensitive enough to detect small changes in ribonucleotide content, although strand-specific Southern blot probing improves the sensitivity. As a measure of one of the most striking biological consequences of ribonucleotides in DNA, spontaneous mutagenesis can be analyzed using a forward mutation assay. Using appropriate reporter genes, rare mutations that results in the loss of function can be selected and overall and specific mutation rates can be measured by combining data from fluctuation experiments with DNA sequencing of the reporter gene. The fluctuation assay is applicable to examine a wide variety of mutagenic processes in specific genetic background or growth conditions.

Treatment of genomic DNA with alkali followed by alkaline gel electrophoresis allows for semi-quantitative detection of the DNA fragmentation due to the abundance of stably incorporated ribonucleotides. Figure 2 shows the gel images of yeast genomic DNA treated with or without KOH5. The M644L variant of Pol2, the catalytic subunit of Pol&#949;, has reduced the ability to incorporate ribonucleotides while the M644G mutant incorporates m...

Watch this Scientific Journal Video about Studying Ribonucleotide Incorporation: Strand-specific Detection of Ribonucleotides in the Yeast Genome and Measuring Ribonucleotide-induced Mutagenesis at Jo

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

Ribonucleotides are among the most abundant non-canonical nucleotides incorporated into the genome during eukaryotic nuclear DNA replication. If not properly removed, ribonucleotides can cause DNA damage and mutagenesis. Here, we present two experimental approaches that are used to assess the abundance of ribonucleotide incorporation into DNA and its mutagenic effects.

The presence of ribonucleotides in nuclear DNA has been shown to be a source of genomic instability. The extent of ribonucleotide incorporation can be ...

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.

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Research

JoVE Journal

Biology

7.6K Views.  National Institute of Environmental Health Sciences, NIH, DHHS. 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 blottin...

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