1. Volume 49, Issue 5, Pages 1010-1015 (March 2013)
Topoisomerase 1-Mediated Removal of Ribonucleotides from Nascent Leading-Strand DNA 
Jessica S. Williams, Dana J. Smith, Lisette Marjavaara, Scott A. Lujan, Andrei Chabes, Thomas A. Kunkel 
Molecular Cell 
Volume 49, Issue 5, Pages (March 2013)
DOI: /j.molcel
Copyright © 2013 Elsevier Inc. Terms and Conditions

2. Figure 1
Top1-Dependent Removal of Genomic Ribonucleotides Incorporated into the Nascent Leading Strand by Pol ε
(A) The orientation of the URA3 reporter with respect to coding sequence is indicated as orientation 1 (OR1) or orientation 2 (OR2). DNA template strands are in black, the nascent leading strand is in blue, and the nascent lagging strand in green. The annealing location of probes A and B are indicated with dotted lines.
(B) Detection of alkali-sensitive sites in yeast genomic DNA by alkaline hydrolysis and Southern analysis using strand-specific radiolabeled probes that anneal to the nascent leading strand. The sizes of DNA markers are shown on the left. All strains harbor the pol2-M644G mutator allele.
(C) The data presented in (B) were quantified to determine the fraction of total alkali-sensitive fragments at each position along the membrane. The radioactive intensity (arbitrary units) was measured at 0.1 mm intervals and divided by the total amount of signal in that lane. The vertical axis corresponds to the DNA marker positions in (B). Curves are derived using data from four independent experiments.
(D) Mean DNA fragment sizes (±SD) were determined using quantitation of the alkali-sensitivity data (Supplemental Experimental Procedures), and p values were calculated using a paired t test. See also Figure S1.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2013 Elsevier Inc. Terms and Conditions

3. Figure 2 S Phase Checkpoint Activation Is Eliminated by TOP1 Deletion
Western blot detection of Rnr3 from whole-cell extracts. Immunoblotting was performed using an antibody to Rnr3 or actin (loading control). Elevation of Rnr3 protein level is an indicator of S phase checkpoint activation (Kumar et al., 2010). All strains harbor the pol2-M644G mutator allele.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2013 Elsevier Inc. Terms and Conditions

4. Figure 3
Top1-Mediated Ribonucleotide Removal Affects Multiple Phenotypes
(A) Flow cytometry analysis. In each histogram, the horizontal axis represents the fluorescence parameter and the vertical axis represents the number of cells. Plotted black lines represent the raw data and the smoothed data generated by ModFit software analysis. The gray shaded areas represent cells in G1 or G2/M phases, and the striped area represents cells in S phase.
(B) The percentage of cells in each stage of the cell cycle was calculated based on flow cytometry analysis of DNA content. The experiment was performed in triplicate, data are displayed as the mean percentage of cells ±SE; ∗p = 0.023, ∗∗p =  (two-tailed Student’s t tests).
(C) Measurement of dNTP pools. Each strain genotype was independently analyzed twice. The data displayed represent the mean total dNTP abundance ±SE; ∗p = (two-tailed Student’s t test). All strains harbor the pol2-M644G mutant allele.
(D) A quantitative HU-survival assay was performed by plating G1-arrested cells onto YPDA agar ± 150 mM HU. The graph represents data for the indicated pol2-M644G mutant strains from three independent experiments with percent survival calculated as the percentage of surviving cells compared to the untreated control (±SE); ∗p <  (two-tailed Student’s t test). See also Figure S2.
(E) Ten-fold serial dilutions of exponentially growing cells spotted onto YPDA agar plates (untreated) or exposed to 150 mM HU.
(F) The specific mutation rate of 2–5 bp deletions in repeat sequences was determined by sequencing a collection of 5-FOA-resistant colonies from each strain. The orientation-dependent difference in mutation rate results from a 2 bp deletion hot spot that is only observed in URA3-OR2. See also Figures S3 and S4.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2013 Elsevier Inc. Terms and Conditions

5. Figure 4 A Model Depicting Pathways of Ribonucleotide Removal from DNA
Ribonucleotides incorporated into genomic DNA by Pol ε are normally repaired during RER. Loss of RNase H2 activity (rnh201Δ) results in unrepaired ribonucleotides that are targets for Top1. Results from this study show that cleavage and removal of ribonucleotides from genomic DNA by Top1 is the cause of several genome-instability phenotypes in yeast that include spontaneous mutagenesis, replicative stress, and checkpoint activation. Genome instability may arise during processing of the “dirty” unligatable DNA ends created by Top1 cleavage, possibly generating DNA nicks, double-strand breaks, and/or recombination. The red triangle indicates the position of the 2′,3′-cyclic phosphate.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2013 Elsevier Inc. Terms and Conditions