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 mg/mL RNase A to the DNA and incubate for 30 min at 37 &#176;C.</li>
		<li>Precipitate the DNA using 0.1 volumes of 3 M sodium acetate (pH 5.2) and 2.5 volumes of 100% ethanol for 20 min on ice.</li>
		<li>Centrifuge at 16,000 x g for 20 min at 4 &#176;C. Remove the supernatant using a pipette.</li>
		<li>Wash the pellet twice with 500 mL of 70% ethanol. Remove the supernatant using a pipette.</li>
		<li>Dry the pellet on bench and resuspend in 35 ml of 1x TE buffer.</li>
		<li>Quantitate the DNA using a fluorimeter (Table of Materials) with the dsDNA BR Assay kit.</li>
		<li>Precipitate 5 mg of DNA from each sample using 0.1 volumes of 3 M sodium acetate, pH 5.2 and 2.5 volumes of 100% EtOH for a total volume of 200 mL (add H2O if necessary, additional 5 mg of DNA is needed if the neutral gel control is necessary). Incubate on ice for 20 min.</li>
		<li>Centrifuge at 16,000 x g for 20 min at 4 &#176;C. Remove the supernatant using a pipette.</li>
		<li>Dry the pellet on bench. Resuspend the DNA in 14 mL of H2O.</li>
	</ol>
	</li>
	<li>Alkaline hydrolysis and gel electrophoresis
	<ol>
		<li>Add 6 mL of 1 M KOH and incubate at 55 &#176;C for 2 h.</li>
		<li>Incubate the samples on ice for 5 min.</li>
		<li>Add 4 mL of 6x alkaline DNA loading buffer: 300 mM KOH, 6 mM EDTA, pH 8.0, 18% polysucrose (Table of Materials), 0.15% bromocresol green, and 0.25% xylene cyanol FF.</li>
		<li>Cast an alkaline gel that is comprised of 1% agarose, 50 mM NaOH, 1 mM EDTA, pH 8.0. Use a comb that allows for 24 mL of DNA sample to be loaded per lane.</li>
		<li>Run the gel at room temperature in 1x alkaline electrophoresis buffer: 50 mM NaOH, 1 mM EDTA, pH 8.0. Include an appropriate DNA size marker.</li>
		<li>Spin down the tubes briefly and load the entire 24 mL sample into the alkaline agarose gel. Electrophorese for 30 min at 30 V, followed by 18 - 20 h at 10 V.</li>
		<li>Neutralize the gel for 2 incubations of 45 min each at room temperature with gentle agitation in Neutralization Buffer I: 1 M Tris-HCl, 1.5 M NaCl.</li>
		<li>Stain the DNA by soaking the neutralized gel in 200 mL of water containing a 1/10,000 dilution of SYBR Gold stain for 2 h at room temperature with gentle shaking.</li>
		<li>Visualize the DNA using a UV transilluminator. Increased alkali-sensitivity causes the appearance of smaller DNA fragments, indicating a higher density of unrepaired ribonucleotides in DNA5.</li>
	</ol>
	</li>
	<li>Southern transfer
	<ol>
		<li>Transfer the DNA from the alkaline gel to the positively charged nylon membrane by capillary action10. An example of how this transfer can be set up is provided in Sambrook and Russell&#8217;s manual10.</li>
		<li>Soak the alkaline gel for 15 min at room temperature in Alkaline Transfer Buffer: 0.4 M NaOH, 1 M NaCl.</li>
		<li>Wet the nylon membrane (cut to the size of the gel) briefly with water, and then soak for 5 min in Alkaline Transfer Buffer.</li>
		<li>Set up the transfer platform by inverting 3 glass beakers (100 mL) in a glass baking dish. This dish should be slightly larger than the size of the agarose gel. Set a glass plate over top of them.</li>
		<li>Drape 2 large pieces of 3 MM CHR chromatography paper (cut to the size of the width of the gel plus longer sides that can drape over the edges into the buffer reservoir) over the glass plate.</li>
		<li>Pour Alkaline Transfer Buffer over the chromatography paper and half fill the glass dish. Smooth out the bubbles using a 5 mL glass pipette.</li>
		<li>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.</li>
		<li>Place the pre-soaked nylon membrane on top of the gel. Smooth out the bubbles.</li>
		<li>Wet 2 pieces of paper cut to the size of the agarose gel. Place them on top of the membrane, and smooth out any bubbles.</li>
		<li>Place a large stack of paper towels (cut to a size slightly larger than the agarose gel) on top of the transfer sandwich. Carefully place a glass plate on top of the paper towels. Add a weighted object to the top (a book with a mass of&#160;400 g works well).</li>
		<li>Let the transfer proceed overnight at room temperature.</li>
		<li>Carefully take the transfer stack apart and soak the membrane for 15 min at room temperature in Neutralization Buffer II: 0.5 M Tris-HCl, pH 7.2, 1 M NaCl.</li>
		<li>Crosslink the DNA to the charged nylon membrane using a UV crosslinker. Place the membrane in the cross-linker and use the autocrosslink setting.</li>
	</ol>
	</li>
	<li>Southern probe preparation 
	Note: The approach described here to detect alkali-sensitive sites in the nascent leading- versus lagging-strand of DNA strand is based on a protocol published previously11. For our experiment, probing is performed at the URA3 reporter gene which has been inserted in one of two orientations (OR1 or OR2) adjacent to the ARS306 replication origin on chromosome III (Figure 3A). The presence of alkali-sensitive sites in yeast genomic DNA is an indicator of ribonucleotide incorporation, allowing the use of ribonucleotides as the biomarkers of DNA polymerase activity12,13,14. This approach, using a target DNA sequence adjacent to the efficient ARS306 origin of replication, should be amenable to other genomic locations and target genes as well.
	<ol>
		<li>PCR-amplify a 520-base pair segment of the URA3 reporter gene using the following primer pair: URA3-F1 (5&#180;-GCTACATATAAGGAACGTGCTGC) and URA3-R1 (5&#180;-CTTTGTCGCTCTTCGCAATGTC).</li>
		<li>Perform the PCR reaction using 10 - 20 ng of yeast genomic DNA as a template, 4 &#956;L of each primer (10 &#956;M stock), 10 &#956;L of 10x Ex Taq Buffer (Mg2+ plus), 8 &#956;L of dNTP mixture (2.5 mM each), 0.5 &#956;L of Ex Taq DNA Polymerase (5 U/&#956;L), and adjust the final volume to 100 &#956;L with H2O.</li>
		<li>Run the following PCR program: 95 &#176;C for 5 min; 30 cycles of 95 &#176;C for 1 min, 55 &#176;C for 1 min, 72 &#176;C for 2 min; 72 &#176;C for 10 min and hold at 4 &#176;C.</li>
		<li>Purify the PCR product using a PCR purification kit and elute the DNA using 50 mL of H2O. This can now be used as the template in the radiolabeling reaction.</li>
		<li>Use the following primers for strand-specific radiolabeling: Use URA3-A (5&#180;-CTCATCCTAGTCCTGTTGCTGCC) for visualizing DNA that anneals to probe A. This corresponds to the nascent leading strand DNA when URA3 is in OR2 and the tnascent lagging strand DNA when URA3 is in OR1. Use URA3-B (5&#180;-CAGTACCCTTAGTATATTCTCCAG) for visualizing nascent DNA that anneals to probe B. This corresponds to the nascent leading strand DNA when URA3 is in OR1 and nascent lagging strand DNA when URA3 is in OR2 (Figure 3).
		<ol>
			<li>Perform the radiolabeling reaction using 10 - 20 ng amplified URA3 fragment as template DNA, 2 &#956;L of primer (10 &#956;M stock), 5 &#956;L of 10x Ex Taq Buffer (Mg2+ plus), 4 &#956;L of 2.5 mM dNTPs (minus dCTP), 5 &#956;L (50 &#956;Ci) of a-32P-dCTP, 0.5 &#956;L of ExTaq DNA Polymerase (5 U/&#956;L) and adjust the final volume to 50 &#956;L with H2O. Run the following program for radiolabeling: 94 &#176;C for 5 min; 25 cycles of 94 &#176;C for 1 min, 55 &#176;C for 30 s, 72 &#176;C for 1 min; 72 &#176;C for 5 min and hold at 4 &#176;C.</li>
		</ol>
		</li>
		<li>Use a G-25 spin column to remove unincorporated radioactive label. Prior to adding the radiolabeled sample, centrifuge the column for 1 min at 720 x g and removethe buffer by pipetting. Load the radiolabeled reaction product and centrifuge for 2 min at 720 x g to elute the labeled probe.</li>
		<li>Incubate at 95 &#176;C for 5 min to denature the probe immediately prior to hybridization. Cool briefly on ice.</li>
	</ol>
	</li>
	<li>Southern Hybridization
	<ol>
		<li>Roll the wetted membrane into a hybridization tube and add 25 mL of freshly prepared Hybridization buffer: 0.5 M sodium phosphate buffer, pH 7.2, 7% sodium dodecyl sulfate (SDS), 1% bovine serum albumin (BSA). Incubate at 65 &#176;C for 1 - 2 h with rotation in a hybridization oven.</li>
		<li>Carefully pour off the solution and add 25 mL of Hybridization buffer plus the radiolabelled probe. Incubate at 65 &#176;C for 16 - 18 h with rotation.</li>
		<li>Pour off the solution into a radioactive waste container. Perform 5 washes of 5 min each at room temperature with Phosphate-SDS Washing Solution I: 40 mM sodium phosphate buffer, pH 7.2, 5% SDS, 0.5% BSA, 1 mM EDTA, pH 8.0.</li>
		<li>Perform 2 washes of 15 min each at 65 &#176;C with Phosphate-SDS Washing Solution II: 40 mM sodium phosphate buffer, pH 7.2, 1% SDS, 1 mM EDTA, pH 8.0.</li>
		<li>Remove the membrane from the hybridization tube using tweezers and place in an opened plastic sleeve protector. Cover and expose to an imaging plate. Try a number of different exposure times ranging from 4 h to 4 d.</li>
		<li>If necessary, strip and re-probe the blot using a different probe approximately 3 - 4 times. To strip, incubate the membrane for 2 h at 65 &#176;C in 25 mL of a 50% formamide, 2x SSPE solution. 
		Note: 20x SSPE stock solution: 3 M NaCl, 0.2 M NaH2PO4, 0.02 M EDTA, pH 7.4.</li>
		<li>Pour off the solution into a radioactive waste container. Perform 2 washes of 15 min each at 65 &#176;C in Phosphate-SDS Washing Solution II.</li>
		<li>Check the membrane with a Geiger counter and repeat the stripping protocol if signal remains.</li>
		<li>Perform pre-hybridization and hybridization as described above.</li>
	</ol>
	</li>
</ol>
2. Measuring Mutation Rate and Specificity in S. cerevisiae Strains
<ol>
	<li>Prepare the cell culture
	<ol>
		<li>Inoculate a single colony (healthy yeast cells takes 2 - 3 days at 30 &#176;C to form proper sized colonies) grown on YPD agar supplemented with 100 mg/mL adenine into 5 mL of YPDA broth supplemented with 250 mg/mL adenine (the supplementation of adenine is only necessary if the strain used is an adenine auxotroph). Grow in a shaker incubator or on a rotator at 30 &#176;C until the culture reaches saturation (36 - 48 h for a strain without a severe growth defect).</li>
		<li>Spin down the cells at ~2000 x g for 5 min.</li>
		<li>Discard the supernatant and wash the cells with 5 mL of sterile water.</li>
		<li>Resuspend the cells in 500 mL of sterile water.</li>
	</ol>
	</li>
	<li>Dilution of the cell culture and plating
	<ol>
		<li>For non-selection controls, dilute the cells in a 96-well plate by a factor of 1,600,000 and plate 100 mL of each culture onto a complete (COM) plate.</li>
		<li>To select for ura3 mutants, plate 100 mL of undiluted cells onto a 5-FOA plate for a wild-type (WT) strain. Dilute the cells accordingly if the mutation rate of the strain of interest is expected to be higher than in a WT strain.</li>
		<li>To select for can1 mutants, dilute the cells 5-fold and plate 100 mL onto a canavanine-containing plate (CAN) for a WT strain. The dilution factor is 0.5.</li>
		<li>Incubate the plates at 30 &#176;C until yeast colonies form. For strains without a growth defect, 2 - 3 days is usually sufficient for COM and CAN plates and 3 - 5 days for 5-FOA plates. To compare the mutation rates between different strains, it is best to choose the same day of growth to count colonies. Store the plates ready to be counted in the cold room (~4 &#176;C).</li>
	</ol>
	</li>
	<li>Calculation of spontaneous mutation rate
	<ol>
		<li>Count visible colonies on each plate and take notes of colony numbers</li>
		<li>Calculate the mutation rate using Drake&#8217;s formula: &#956; = f &#247; ln (&#956;Nt). f is the mutation frequency calculated by number of mutants divided by number of cells in the final culture. Nt is the number of cells in the final culture and&#160;&#956; is the mutation rate. This equation can be solved by an iterative method such as the Newton-Raphson method. There are other estimator methods, including the Lea and Coulson test, to calculate the mutation rate15.</li>
		<li>Calculate the confidence interval assuming log normal distribution of the utation rates of individual isolates. The 95% confidence interval is most often used.</li>
	</ol>
	</li>
	<li>Analysis of mutation spectrum
	<ol>
		<li>Pick a single colony from each 5-FOA or CAN plate and refresh on a YPDA plate.</li>
		<li>Perform colony PCR to amplify the URA3 or CAN1 gene and sequence in order to detect mutations using the following primers: URA3-F (5&#8217;-CGCATATGTGGTGTTGAAGAA-3&#8217;) and URA3-R (5&#8217;-TGTGAGTTTAGTATACATGCA-3&#8217;) for both PCR amplification and sequencing of the 800 bp URA3 gene; CAN1-F (5&#8217;-CTTCTACTCCGTCTGCTTTC-3&#8217;) and CAN1-R (5&#8217;-CAGAGTTCTTCAGACTTC-3&#8217;) for amplification and CAN1-AR (5&#8217;- TGAAATGTGAAGGCAGCGTT-3&#8217;), CAN1-9R (5&#8217;- CCTGCAACACCAGTGATA-3&#8217;), CAN1-10R (5&#8217;- GAGGATGTAACAGGGATGAAT-3&#8217;) and CAN1-BR (5&#8217;-CGGTGTATGACTTATGAGGG-3&#8217;) for sequencing of the 1800 bp CAN1 reporter gene.</li>
		<li>To perform colony PCR, resuspend a single colony in 5 &#956;L of H2O and use 1 &#956;L as template. Add 1 &#956;L of each of the primers (5 &#956;M stock), 2 &#956;L of 5x KAPA2G Buffer (Mg2+ plus), 0.5 &#956;L of dNTP (2.5 mM), 0.5 &#956;L of KAPA2G Fast DNA Polymerase, 12 &#956;L of H2O.</li>
		<li>For the amplification of URA3 gene, run the following PCR program: 95 &#176;C for 5 min; 5 cycles of 95 &#176;C for 10 s, 61 &#176;C for 15 s, 72 &#176;C for 30 s; 35 cycles of 95 &#176;C for 10 s, 61 &#176;C for 15 s, 72 &#176;C for 30 s; 72 &#176;C for 2 min and hold at 4 &#176;C. For the amplification of CAN1 gene, run the following PCR program: 95 &#176;C for 5 min; 5 cycles of 95 &#176;C for 10 s, 61 &#176;C for 15 s, 72 &#176;C for 45 s; 35 cycles of 95 &#176;C for 10 s, 61 &#176;C for 15 s, 72 &#176;C for 45 s; 72 &#176;C for 2 min and hold at 4 &#176;C.</li>
		<li>Align the sequencing results to a reference sequence using a program such as DNASTAR Seqman Pro or Sequencher to identify mutations.</li>
		<li>Compile the mutations data by mutation type (Figure 4) or mutation location (Figure 5). Calculate the specific mutation rates by the frequency of a mutation (the number of events observed divided by number of mutants sequenced) multiplied by the overall mutation rate.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

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 H&lt;sub&gt;2&lt;/sub&gt;O 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 H&lt;sub&gt;2&lt;/sub&gt;O up to 1 L. Adjust pH to 5.8. Autoclave and 30 &amp;ndash; 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 &amp;deg;C and cool down to 56 &amp;deg;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 &amp;deg;C and cool down to 60 &amp;deg;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 &amp;deg;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>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; (S9638);&lt;br/&gt; Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt; (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...

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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 ...

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Research

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Biology

7.7K Views.  National Institute of Environmental Health Sciences, NIH, DHHS. 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.

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

Genome-wide Quantification of Translation in Budding Yeast by Ribosome Profiling

Translation of mRNA into proteins is a complex process involving several layers of regulation. It is often assumed that changes in mRNA transcription reflect changes in protein synthesis, but many exceptions have been observed. Recently, a technique called ribosome profiling (or Ribo-Seq) has emerged as a powerful method that allows identification, with high accuracy, which regions of mRNA are translated into proteins and quantification of translation at the genome-wide level. Here, we present a generalized protocol for genome-wide quantification of translation using Ribo-Seq in budding yeast. In addition, combining Ribo-Seq data with mRNA abundance measurements allows us to simultaneously quantify translation efficiency of thousands of mRNA transcripts in the same sample and compare changes in these parameters in response to experimental manipulations or in different physiological states. We describe a detailed protocol for generation of ribosome footprints using nuclease digestion, isolation of intact ribosome-footprint complexes via sucrose gradient fractionation, and preparation of DNA libraries for deep sequencing along with appropriate quality controls necessary to ensure accurate analysis of in vivo translation.

Translational regulation plays an important role in the control of protein abundance. Here, we describe a high-throughput method for quantitative analysis of translation in the budding yeast Saccharomyces cerevisiae.

Translation of mRNA into proteins is a complex process involving several layers of regulation. It is often assumed that changes in mRNA transcription ...

Biochemistry

Single Molecule Fluorescence In Situ Hybridization (smFISH) Analysis in Budding Yeast Vegetative Growth and Meiosis

Single molecule fluorescence in situ hybridization (smFISH) is a powerful technique to study gene expression in single cells due to its ability to detect and count individual RNA molecules. Complementary to deep sequencing-based methods, smFISH provides information about the cell-to-cell variation in transcript abundance and the subcellular localization of a given RNA. Recently, we have used smFISH to study the expression of the gene&#160;NDC80&#160;during meiosis in budding yeast, in which two transcript isoforms exist and the short transcript isoform has its entire sequence shared with the long isoform. To confidently identify each transcript isoform, we optimized known smFISH protocols and obtained high consistency and quality of smFISH data for the samples acquired during budding yeast meiosis. Here, we describe this optimized protocol, the criteria that we use to determine whether high quality of smFISH data is obtained, and some tips for implementing this protocol in&#160;other yeast strains and growth conditions.

Single Molecule Fluorescence In Situ Hybridization smFISH Analysis in Budding Yeast Vegetative Growth and Meiosis

This single molecule fluorescence in situ hybridization protocol is optimized to quantify the number of RNA molecules in budding yeast during vegetative growth and meiosis.

Single molecule fluorescence in situ hybridization (smFISH) is a powerful technique to study gene expression in single cells due to its ability to detect ...

Genetics

Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity

Global defects in RNA polymerase II transcription might be overlooked by transcriptomic studies analyzing steady-state RNA. Indeed, the global decrease in mRNA synthesis has been shown to be compensated by a simultaneous decrease in mRNA degradation to restore normal steady-state levels. Hence, the genome-wide quantification of mRNA synthesis, independently from mRNA decay, is the best direct reflection of RNA polymerase II transcriptional activity. Here, we discuss a method using non-perturbing metabolic labeling of nascent RNAs in Saccharomyces cerevisiae (S. cerevisiae). Specifically, the cells are cultured for 6 min with a uracil analog, 4-thiouracil, and the labeled newly transcribed RNAs are purified and quantified to determine the synthesis rates of all individual mRNA. Moreover, using labeled Schizosaccharomyces pombe cells as internal standard allows comparing mRNA synthesis in different S. cerevisiae strains. Using this protocol and fitting the data with a dynamic kinetic model, the corresponding mRNA decay rates can be determined.

Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity

The protocol described here is based on the genome-wide quantification of newly synthesized mRNA purified from yeast cells labeled with 4-thiouracil. This method allows to measure mRNA synthesis uncoupled from mRNA decay and, thus, provides an accurate measurement of RNA polymerase II transcription.

Global defects in RNA polymerase II transcription might be overlooked by transcriptomic studies analyzing steady-state RNA. Indeed, the global decrease in ...

ACT1-CUP1 Assays Determine the Substrate-Specific Sensitivities of Spliceosomal Mutants in Budding Yeast

Mutations introduced in the spliceosome or its substrate have significantly contributed to our understanding of the intricacies of spliceosomal function. Whether disease-related or functionally selected, many of these mutations have been studied using growth assays in the model organism Saccharomyces cerevisiae (yeast). The splicing-specific copper growth assay, or ACT1-CUP1 assay, provides a comprehensive analysis of mutation at the phenotypic level. The ACT1-CUP1 assay utilizes reporters that confer copper tolerance when correctly spliced. Thus, in the presence of copper, changes in yeast viability correlate to changes in mRNA production through splicing. In a typical experiment, the yeast spliceosome is challenged with different non-consensus splicing reporters and the splicing factor mutation of interest to detect any synergetic or antithetical impact on splicing. Here a full description of copper plate preparation, plating of yeast cells, and data evaluation are given. A selection of complimentary experiments is described, highlighting the versatility of the ACT1-CUP1 reporters. The ACT1-CUP1 assay is a handy tool in the splicing toolbox thanks to the direct read-out of mutational effect(s) and the comparative possibilities from the continuing use in the field.

The ACT1-CUP1 assay, a copper growth assay, provides a quick readout of precursor messenger RNA (pre-mRNA) splicing and the impact mutant splicing factors have on spliceosomal function. This study provides a protocol and highlights the customization possible to address the splicing question of interest.

Mutations introduced in the spliceosome or its substrate have significantly contributed to our understanding of the intricacies of spliceosomal function. ...

Monitoring Protein-RNA Interaction Dynamics In Vivo at High Temporal Resolution Using &#967;CRAC

The interaction between RNA-binding proteins (RBPs) and their RNA substrates exhibits fluidity and complexity. Within its lifespan, a single RNA can be bound by many different RBPs that will regulate its production, stability, activity, and degradation. As such, much has been done to understand the dynamics that exist between these two types of molecules. A particularly important breakthrough came with the emergence of &#8216;cross-linking and immunoprecipitation&#8217; (CLIP). This technique allowed stringent investigation into which RNAs are bound by a particular RBP. In short, the protein of interest is UV cross-linked to its RNA substrates in vivo, purified under highly stringent conditions, and then the RNAs covalently cross-linked to the protein are converted into cDNA libraries and sequenced. Since its conception, many derivative techniques have been developed in order to make CLIP amenable to particular fields of study. However, cross-linking using ultraviolet light is notoriously inefficient. This results in extended exposure times that make the temporal study of RBP-RNA interactions impossible. To overcome this issue, we recently designed and built much-improved UV irradiation and cell harvesting devices. Using these new tools, we developed a protocol for time-resolved analyses of RBP-RNA interactions in living cells at high temporal resolution: Kinetic CRoss-linking and Analysis of cDNAs (&#967;CRAC). We recently used this technique to study the role of yeast RBPs in nutrient stress adaptation. This manuscript provides a detailed overview of the &#967;CRAC method and presents recent results obtained with the Nrd1 RBP.

Kinetic cross-linking and analysis of cDNA is a method that allows investigation of the dynamics of protein-RNA interactions in living cells at high temporal resolution. Here the protocol is described in detail, including the growth of yeast cells, UV cross-linking, harvesting, protein purification, and next generation sequencing library preparation steps.

The interaction between RNA-binding proteins (RBPs) and their RNA substrates exhibits fluidity and complexity. Within its lifespan, a single RNA can be ...

Nonradioactive Assay to Measure Polynucleotide Phosphorylation of Small Nucleotide Substrates

Polynucleotide kinases (PNKs) are enzymes that catalyze the phosphorylation of the 5&#39; hydroxyl end of DNA and RNA oligonucleotides. The activity of PNKs can be quantified using direct or indirect approaches. Presented here is a direct, in vitro approach to measure PNK activity that relies on a fluorescently-labeled oligonucleotide substrate and polyacrylamide gel electrophoresis. This approach provides resolution of the phosphorylated products while avoiding the use of radiolabeled substrates. The protocol details how to set up the phosphorylation reaction, prepare and run large polyacrylamide gels, and quantify the reaction products. The most technically challenging part of this assay is pouring and running the large polyacrylamide gels; thus, important details to overcome common difficulties are provided. This protocol was optimized for Grc3, a PNK that assembles into an obligate pre-ribosomal RNA processing complex with its binding partner, the Las1 nuclease. However, this protocol can be adapted to measure the activity of other PNK enzymes. Moreover, this assay can also be modified to determine the effects of different components of the reaction, such as the nucleoside triphosphate, metal ions, and oligonucleotides.

This protocol describes a nonradioactive assay to measure kinase activity of polynucleotide kinases (PNKs) on small DNA and RNA substrates.

Polynucleotide kinases (PNKs) are enzymes that catalyze the phosphorylation of the 5&#39; hydroxyl end of DNA and RNA oligonucleotides. The activity of ...

DNAzyme-dependent Analysis of rRNA 2&#8217;-O-Methylation

Guide box C/D small nucleolar RNAs (snoRNAs) catalyze 2&#8217;-O-methylation of ribosomal and small nuclear RNA. However, a large number of snoRNA in higher eukaryotes may promiscuously recognize other RNA species and 2&#8217;-O-methylate multiple targets. Here, we provide step-by-step guide for the fast and non-expensive analysis of the site-specific 2&#8217;-O-methylation using a well-established method employing short DNA oligonucleotides called DNAzymes. These DNA fragments contain catalytic sequences which cleave RNA at specific consensus positions, as well as variable homology arms directing DNAzyme to its RNA targets. DNAzyme activity is inhibited by 2-&#8217;O-methylation of the nucleotide adjacent to the cleavage site in the RNA. Thus, DNAzymes, limited only by the consensus of the cleaved sequence, are perfect tools for the quick analysis of snoRNA-mediated RNA 2&#8217;-O-methylation. We analyzed snoRNA snR13- and snR47-guided 2&#8217;-O-methylation of 25S ribosomal RNA in Saccharomyces cerevisiae to demonstrate the simplicity of the technique and to provide a detailed protocol for the DNAzyme-dependent assay.

Here we present a protocol for DNAzyme-dependent cleavage of RNA. This enables fast and site-dependent analysis of RNA 2&#8217;-O-methylation. This approach can be used for the preliminary or major assessment of snoRNA activity.

Guide box C/D small nucleolar RNAs (snoRNAs) catalyze 2&#8217;-O-methylation of ribosomal and small nuclear RNA. However, a large number of snoRNA in ...

Developmental Biology

Polysome Profiling without Gradient Makers or Fractionation Systems

Polysome fractionation by sucrose density gradient centrifugation is a powerful tool that can be used to create ribosome profiles, identify specific mRNAs being translated by ribosomes, and analyze polysome associated factors. While automated gradient makers and gradient fractionation systems are commonly used with this technique, these systems are generally expensive and can be cost-prohibitive for laboratories that have limited resources or cannot justify the expense due to their infrequent or occasional need to perform this method for their research. Here, a protocol is presented to reproducibly generate polysome profiles using standard equipment available in most molecular biology laboratories without specialized fractionation instruments. Moreover, a comparison of polysome profiles generated with and without a gradient fractionation system is provided. Strategies to optimize and produce reproducible polysome profiles are discussed. Saccharomyces cerevisiae is utilized as a model organism in this protocol. However, this protocol can be easily modified and adapted to generate ribosome profiles for many different organisms and cell types.

This protocol describes how to generate a polysome profile without using automated gradient makers or gradient fractionation systems.

Polysome fractionation by sucrose density gradient centrifugation is a powerful tool that can be used to create ribosome profiles, identify specific mRNAs ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Simultaneous Mapping and Quantitation of Ribonucleotides in Human Mitochondrial DNA

Established approaches to estimate the number of ribonucleotides present in a genome are limited to the quantitation of incorporated ribonucleotides using short synthetic DNA fragments or plasmids as templates and then extrapolating the results to the whole genome. Alternatively, the number of ribonucleotides present in a genome may be estimated using alkaline gels or Southern blots. More recent in vivo approaches employ Next-generation sequencing allowing genome-wide mapping of ribonucleotides, providing the position and identity of embedded ribonucleotides. However, they do not allow quantitation of the number of ribonucleotides which are incorporated into a genome. Here we describe how to simultaneously map and quantitate the number of ribonucleotides which are incorporated into human mitochondrial DNA in vivo by Next-generation sequencing. We use highly intact DNA and introduce sequence specific double strand breaks by digesting it with an endonuclease, subsequently hydrolyzing incorporated ribonucleotides with alkali. The generated ends are ligated with adapters and these ends are sequenced on a Next-generation sequencing machine. The absolute number of ribonucleotides can be calculated as the number of reads outside the recognition site per average number of reads at the recognition site for the sequence specific endonuclease. This protocol may also be utilized to map and quantitate free nicks in DNA and allows adaption to map other DNA lesions that can be processed to 5&#180;-OH ends or 5&#180;-phosphate ends. Furthermore, this method can be applied to any organism, given that a suitable reference genome is available. This protocol therefore provides an important tool to study DNA replication, 5&#180;-end processing, DNA damage, and DNA repair.

Here we describe a method amenable to simultaneously quantitate and genome-wide map ribonucleotides in highly intact DNA at single-nucleotide resolution, combining enzymatic cleavage of genomic DNA with its alkaline hydrolysis and subsequent 5&#180;-end sequencing.

Established approaches to estimate the number of ribonucleotides present in a genome are limited to the quantitation of incorporated ribonucleotides using ...

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

Summary

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Chapters in this video

Genome-wide Quantification of Translation in Budding Yeast by Ribosome Profiling

Single Molecule Fluorescence In Situ Hybridization (smFISH) Analysis in Budding Yeast Vegetative Growth and Meiosis

Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity

ACT1-CUP1 Assays Determine the Substrate-Specific Sensitivities of Spliceosomal Mutants in Budding Yeast

Monitoring Protein-RNA Interaction Dynamics In Vivo at High Temporal Resolution Using χCRAC

Nonradioactive Assay to Measure Polynucleotide Phosphorylation of Small Nucleotide Substrates

DNAzyme-dependent Analysis of rRNA 2’-O-Methylation

Polysome Profiling without Gradient Makers or Fractionation Systems

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

Simultaneous Mapping and Quantitation of Ribonucleotides in Human Mitochondrial DNA