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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</ol>

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 ...

studying-ribonucleotide-incorporation-strand-specific-detection

Research

JoVE Journal

Biology

7.7K Views.  National Institute of Environmental Health Sciences, NIH, DHHS. Ribonucleotides behoren tot de meest voorkomende niet-canonieke nucleotiden in het genoom opgenomen tijdens de eukaryote nucleair DNA-replicatie. Als het niet correct is verwijderd, kan ribonucleotides DNA-beschadiging en mutagenese veroorzaken. Hier presenteren we twee experimentele benaderingen die worden gebruikt voor het beoordelen van de overvloed van ribonucleotide opneming in DNA en zijn mutagene effecten.

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

Gene-targeted Random Mutagenesis to Select Heterochromatin-destabilizing Proteasome Mutants in Fission Yeast

Random mutagenesis of a target gene is commonly used to identify mutations that yield the desired phenotype. Of the methods that may be used to achieve random mutagenesis, error-prone PCR is a convenient and efficient strategy for generating a diverse pool of mutants (i.e., a mutant library). Error-prone PCR is the method of choice when a researcher seeks to mutate a pre-defined region, such as the coding region of a gene while leaving other genomic regions unaffected. After the mutant library is amplified by error-prone PCR, it must be cloned into a suitable plasmid. The size of the library generated by error-prone PCR is constrained by the efficiency of the cloning step. However, in the fission yeast, Schizosaccharomyces pombe, the cloning step can be replaced by the use of a highly efficient one-step fusion PCR to generate constructs for transformation. Mutants of desired phenotypes may then be selected using appropriate reporters. Here, we describe this strategy in detail, taking as an example, a reporter inserted at centromeric heterochromatin.

This article describes a detailed methodology for random mutagenesis of a target gene in fission yeast. As an example, we target rpt4+, which encodes a subunit of the 19S proteasome, and screen for mutations that destabilize heterochromatin.

Random mutagenesis of a target gene is commonly used to identify mutations that yield the desired phenotype. Of the methods that may be used to achieve ...

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 ...

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System 

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers. Genetic studies in mammalian systems have been limited due to the lack of readily available tools including defined mutant genetic cell lines, regulatory expression systems, and appropriate selectable markers. To circumvent these difficulties, model genetic systems in lower eukaryotes have become an attractive choice for the study of functionally conserved DNA repair proteins and pathways. We have developed a model yeast system to study the poorly defined genetic functions of the Werner syndrome helicase-nuclease (WRN) in nucleic acid metabolism. Cellular phenotypes associated with defined genetic mutant backgrounds can be investigated to clarify the cellular and molecular functions of WRN through its catalytic activities and protein interactions. The human WRN gene and associated variants, cloned into DNA plasmids for expression in yeast, can be placed under the control of a regulatory plasmid element. The expression construct can then be transformed into the appropriate yeast mutant background, and genetic function assayed by a variety of methodologies. Using this approach, we determined that WRN, like its related RecQ family members BLM and Sgs1, operates in a Top3-dependent pathway that is likely to be important for genomic stability. This is described in our recent publication [1] at <a href="http://www.impactaging.com">www.impactaging.com</a>. Detailed methods of specific assays for genetic complementation studies in yeast are provided in this paper.

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System

Genetic studies in yeast can be employed to investigate the molecular and cellular functions of human genes in cellular DNA metabolism. Methods are described for the genetic characterization of the human WRN gene product defective in the premature aging disorder Werner syndrome in functionally conserved pathways using yeast as a tractable model system.

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers.  Genetic studies in mammalian ...

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

Combining Magnetic Sorting of Mother Cells and Fluctuation Tests to Analyze Genome Instability During Mitotic Cell Aging in Saccharomyces cerevisiae

Saccharomyces cerevisiae has been an excellent model system for examining mechanisms and consequences of genome instability. Information gained from this yeast model is relevant to many organisms, including humans, since DNA repair and DNA damage response factors are well conserved across diverse species. However, S. cerevisiae has not yet been used to fully address whether the rate of accumulating mutations changes with increasing replicative (mitotic) age due to technical constraints. For instance, measurements of yeast replicative lifespan through micromanipulation involve very small populations of cells, which prohibit detection of rare mutations. Genetic methods to enrich for mother cells in populations by inducing death of daughter cells have been developed, but population sizes are still limited by the frequency with which random mutations that compromise the selection systems occur. The current protocol takes advantage of magnetic sorting of surface-labeled yeast mother cells to obtain large enough populations of aging mother cells to quantify rare mutations through phenotypic selections. Mutation rates, measured through fluctuation tests, and mutation frequencies are first established for young cells and used to predict the frequency of mutations in mother cells of various replicative ages. Mutation frequencies are then determined for sorted mother cells, and the age of the mother cells is determined using flow cytometry by staining with a fluorescent reagent that detects bud scars formed on their cell surfaces during cell division. Comparison of predicted mutation frequencies based on the number of cell divisions to the frequencies experimentally observed for mother cells of a given replicative age can then identify whether there are age-related changes in the rate of accumulating mutations. Variations of this basic protocol provide the means to investigate the influence of alterations in specific gene functions or specific environmental conditions on mutation accumulation to address mechanisms underlying genome instability during replicative aging.

Combining Magnetic Sorting of Mother Cells and Fluctuation Tests to Analyze Genome Instability During Mitotic Cell Aging in Saccharomyces cerevisiae

Mutation rates in young Saccharomyces cerevisiae cells measured through fluctuation tests are used to predict mutation frequencies for mother cells of different replicative ages. Magnetic sorting and flow cytometry are then used to measure actual mutation frequencies and age of mother cells to identify any deviations from predicted mutation frequencies.

Saccharomyces cerevisiae has been an excellent model system for examining mechanisms and consequences of genome instability. Information gained from this ...

Measurement of mRNA Decay Rates in Saccharomyces cerevisiae Using rpb1-1 Strains

mRNA steady state levels vary depending on environmental conditions. Regulation of the steady state accumulation levels of an mRNA ensures that the correct amount of protein is synthesized for the cell&#8217;s specific growth conditions. One approach for measuring mRNA decay rates is inhibiting transcription and subsequently monitoring the disappearance of the already present mRNA. The rate of mRNA decay can then be quantified, and an accurate half-life can be determined utilizing several techniques. In S. cerevisiae, protocols that measure mRNA half-lives have been developed and include inhibiting transcription of mRNA using strains that harbor a temperature sensitive allele of RNA polymerase II, rpb1-1. Other techniques for measuring mRNA half-lives include inhibiting transcription with transcriptional inhibitors such as thiolutin or 1,10-phenanthroline, or alternatively, by utilizing mRNAs that are under the control of a regulatable promoter such as the galactose inducible promoter and the TET-off system. Here, we describe measurement of S. cerevisiae mRNA decay rates using the temperature sensitive allele of RNA polymerase II. This technique can be used to measure mRNA decay rates of individual mRNAs or genome-wide.

Measurement of mRNA Decay Rates in Saccharomyces cerevisiae Using rpb1-1 Strains

The steady state level of specific mRNAs is determined by the rate of synthesis and decay of the mRNA. Genome-wide mRNA degradation rates or the decay rates of specific mRNAs can be measured by determining mRNA half-lives. This protocol focuses on measurement of mRNA decay rates in Saccharomyces cerevisiae.

mRNA steady state levels vary depending on environmental conditions. Regulation of the steady state accumulation levels of an mRNA ensures that the ...

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Determining the mode of action of bioactive chemicals is of interest to a broad range of academic, pharmaceutical, and industrial scientists. Saccharomyces cerevisiae, or budding yeast, is a model eukaryote for which a complete collection of ~6,000 gene deletion mutants and hypomorphic essential gene mutants are commercially available. These collections of mutants can be used to systematically detect chemical-gene interactions, i.e. genes necessary to tolerate a chemical. This information, in turn, reports on the likely mode of action of the compound. Here we describe a protocol for the rapid identification of chemical-genetic interactions in budding yeast. We demonstrate the method using the chemotherapeutic agent 5-fluorouracil (5-FU), which has a well-defined mechanism of action. Our results show that the nuclear TRAMP RNA exosome and DNA repair enzymes are needed for proliferation in the presence of 5-FU, which is consistent with previous microarray based bar-coding chemical genetic approaches and the knowledge that 5-FU adversely affects both RNA and DNA metabolism. The required validation protocols of these high-throughput screens are also described.

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Here we present a cost-effective method for defining chemical-genetic interactions in budding yeast. The approach is built on fundamental techniques in yeast molecular biology and is well suited for the mechanistic interrogation of small to medium collections of chemicals and other media environments.

Determining the mode of action of bioactive chemicals is of interest to a broad range of academic, pharmaceutical, and industrial scientists. ...

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 ...

Determination of S-Phase Duration Using 5-Ethynyl-2'-deoxyuridine Incorporation in Saccharomyces cerevisiae

Eukaryotic DNA replication is a highly regulated process that ensures that the genetic blueprint of a cell is correctly duplicated prior to chromosome segregation. As DNA synthesis defects underlie chromosome rearrangements, monitoring DNA replication has become essential to understand the basis of genome instability. Saccharomyces cerevisiae is a classical model to study cell cycle regulation, but key DNA replication parameters, such as the fraction of cells in the S phase or the S-phase duration, are still difficult to determine. This protocol uses short and non-toxic pulses of 5-ethynyl-2'-deoxyuridine (EdU), a thymidine analog, in engineered TK-hENT1 yeast cells, followed by its detection by Click reaction to allow the visualization and quantification of DNA replication with high spatial and temporal resolution at both the single-cell and population levels by microscopy and flow cytometry. This method may identify previously overlooked defects in the S phase and cell cycle progression of yeast mutants, thereby allowing the characterization of new players essential for ensuring genome stability.

Determination of S-Phase Duration Using 5-Ethynyl-2'-deoxyuridine Incorporation in Saccharomyces cerevisiae

We describe two complementary protocols to accurately determine S-phase duration in S. cerevisiae using EdU, a thymidine analog, which is incorporated in vivo and detected using Click chemistry by microscopy and flow cytometry. It allows for the easy characterization of the duration of DNA replication and overlooked replication defects in mutants.

Eukaryotic DNA replication is a highly regulated process that ensures that the genetic blueprint of a cell is correctly duplicated prior to chromosome ...

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

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