Topoisomerase 1-mediated removal of ribonucleotides from nascent leading-strand DNA

Abstract

RNase H2-dependent ribonucleotide excision repair (RER) removes ribonucleotides incorporated during DNA replication. When RER is defective, ribonucleotides in the nascent leading strand of the yeast genome are associated with replication stress and genome instability. Here, we provide evidence that topoisomerase 1 (Top1) initiates an independent form of repair to remove ribonucleotides from genomic DNA. This Top1-dependent process activates the S phase checkpoint. Deleting TOP1 reverses this checkpoint activation and also relieves replication stress and genome instability in RER-defective cells. The results reveal an additional removal pathway for a very common lesion in DNA, and they imply that the "dirty" DNA ends created when Top1 incises ribonucleotides in DNA are responsible for the adverse consequences of ribonucleotides in RNase H2-defective cells.

Figure 1. Top1-dependent Removal of Genomic Ribonucleotides Incorporated into the Nascent Leading Strand by Polε

(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 part B was 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 part B. Curves are derived using data from four independent experiments. (D) Mean DNA fragment sizes (± standard deviation) were determined using quantitation of the alkali-sensitivity data (Supplemental Experimental Procedures). P-values were calculated using a paired t test.

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.

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, the striped area represent 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 % cells ± standard error. *, P = 0.023, **, P = 0.0136 (two-tailed Student’s t tests). (C) Measurement of dNTP pools. Each strain genotype was independently analyzed twice. The data displayed represents the mean total dNTP abundance ± standard error. *, P = 0.0032 (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 % survival calculated as the percentage of surviving cells compared to the untreated control (± standard error). *, P < 0.0001 (two-tailed Student’s t test). (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-base deletion hotspot that is only observed in URA3-OR2.

Figure 4. A Model Depicting Pathways of Ribonucleotide Removal from DNA

Ribonucleotides incorporated into genomic DNA by Pol εare 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 (DSBs) and/or recombination. The red triangle indicates the position of the 2′,3′-cyclic phosphate.