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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 I (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.
DNA polymerases exclude ribonucleoside triphosphates (rNTPs) from being incorporated into DNA (Joyce, 1997), but this exclusion is imperfect and complicated by the fact that rNTP concentrations in Saccharomyces cerevisiae are much higher than dNTP concentrations (Nick McElhinny et al., 2010b). As a result, in reactions containing rNTP and dNTP concentrations measured in vivo, yeast replicative DNA polymerases incorporate large numbers of ribonucleotides into DNA during synthesis in vitro (Nick McElhinny et al., 2010b). Ribonucleotides are also incorporated into DNA during replication in vivo, both by wild type yeast DNA Polymerase ε (Pol ε) and even more by a variant of Pol ε that contains a Met644 to Gly substitution in the polymerase active site (pol2-M644G; (Nick McElhinny et al., 2010a). These ribonucleotides are normally removed from DNA by Ribonucleotide Excision Repair (RER) (see (Sparks et al., 2012) and references therein), which is initiated when RNase H2 incises the DNA backbone on the 5′-side of the ribonucleotide (Eder and Walder, 1991; Eder et al., 1993; Rydberg and Game, 2002). Fen1 then incises 3′ of the ribonucleotide to release it (Rydberg and Game, 2002), leaving a single nucleotide gap to be filled in by Pol δ (Sparks et al., 2012). The resulting nick contains ligatable DNA ends, i.e., a 5′-phosphate and a 3′-hydroxyl, which can be sealed by DNA ligase 1 to complete RER.
A defect in RER due to deletion of the RNH201 gene encoding the catalytic subunit of RNase H2 leaves unrepaired ribonucleotides in yeast and mouse nuclear genomes that can be detected as alkali-sensitive sites (Lujan et al., 2012; Miyabe et al., 2011; Nick McElhinny et al., 2010a; Reijns et al., 2012). In both a S. cerevisiae pol2-M644G rnh201Δ mutant (Lujan et al., 2012) and a Schizosaccharomyces pombe cdc20-M630F rnh201Δ mutant (Miyabe et al., 2011), these unrepaired ribonucleotides are preferentially incorporated into the nascent leading strand. These data support mutagenesis studies indicating that Pol ε is the primary leading strand replicase for the nuclear genome (Nick McElhinny et al., 2008; Pursell et al., 2007). In order to focus on ribonucleotides incorporated into DNA by the polymerases rather than by the RNA primase associated with Pol α during initiation of lagging strand replication, the current study addresses the biological consequences of failure to remove ribonucleotides incorporated into DNA during leading strand synthesis by Pol ε. pol2-M644G rnh201Δ cells progress slowly through S phase, have slightly elevated dNTP pools and strongly elevated rates of 2-5 base pair deletions in tandemly repeated DNA sequences (Clark et al., 2011; Nick McElhinny et al., 2010a), and they are sensitive to treatment with the replication inhibitor, hydroxyurea (HU) (Lazzaro et al., 2012). The rate of 2-5 base pair deletions in repetitive sequences is also elevated in RNase H2-defective yeast strains encoding wild type Pol ε(Chen et al., 2000; Clark et al., 2011; Kim et al., 2011; Qiu et al., 1999), indicating that wild type Pol ε normally incorporates ribonucleotides during replication that are mutagenic if not repaired.
The discovery that ribonucleotide-dependent mutagenesis is unaffected by loss of DNA mismatch repair (MMR; (Clark et al., 2011) led to the hypothesis that the misaligned mismatches responsible for the 2-5 base pair deletions in RNase H2-deficient strains were generated during ribonucleotide processing outside the context of replication. One possible mechanism was suggested by the discovery that 2-5 base deletions in repetitive sequences associated with transcription depend on Topoisomerase 1 (Top1) (Lippert et al., 2011; Takahashi et al., 2011), an enzyme important for removing replication and transcription-associated supercoils (Wang, 2002). In addition to this function, both vaccinia and human Top1 can also act as endonucleases when a ribonucleotide is present at the cleavage site (Sekiguchi and Shuman, 1997). In this reaction, the 3′-phosphotyrosyl linkage that forms between Top1 and the ribonucleotide is subject to nucleophilic attack by the 2′-OH of the ribose. This releases Top1 to generate “dirty” 5′-OH and 2′,3′-cyclic phosphate ends that must be subsequently processed to permit ligation. Collectively, these studies suggested that the deletions resulting from unrepaired ribonucleotides in RNase H2-deficient strains might depend on the endonuclease activity of Top1. We first tested this possibility in rnh201Δ yeast strains encoding wild type DNA polymerases. As predicted by the hypothesis, the deletion rates in these strains were reduced when TOP1 was deleted (Kim et al., 2011). In addition, biochemical studies in vitro demonstrated that recombinant human Top1 can efficiently incise duplex DNA at sites where ribonucleotides are present (Kim et al., 2011). This led to a model wherein Top1 cleaves unrepaired ribonucleotides in the genome of an RNase H2-defective yeast strain to create DNA ends that must be processed to allow ligation. When these nicks are made in repetitive sequences, end processing provides the opportunity for misaligned intermediates to form that ultimately yield deletions.
This model for generating rare deletions occurring at rates of 10−7 to 10−6 motivated the present study, which addresses two questions. To what extent, if at all, does Top1 endonuclease activity contribute to removing ribonucleotides from the genome of strains lacking RNase H2-dependent RER? Given that Top1 endonuclease creates “dirty” DNA ends that could be problematic for cells, do the genome instability phenotypes of a pol2-M644G rnh201Δ mutant depend on Top1 nicking at unrepaired ribonucleotides in the nascent leading strand of the yeast genome? Here we provide evidence in vivo that yeast Top1 is indeed partially redundant with RNase H2 in removing ribonucleotides from DNA, thereby highlighting a largely unexplored DNA repair process. We also show that constitutive S-phase checkpoint activation, replicative stress and genome instability in pol2-M644G rnh201Δ cells containing unrepaired ribonucleotides in the nascent leading strand are all Top1-dependent, thereby implying that the “dirty” DNA ends created when Top1 incises ribonucleotides in DNA are responsible for these phenotypes.
We began the present study by asking if yeast Top1 is involved in ribonucleotide removal in vivo. Because the 2′-OH on a ribose renders the DNA backbone susceptible to cleavage in alkali, ribonucleotides can be detected by incubating genomic DNA in 0.3 M KOH, and then monitoring the resulting fragments by alkaline-agarose gel electrophoresis followed by Southern blotting using a radiolabeled probe specific for the URA3 reporter gene (Figure 1A). Using this approach and a probe for nascent leading strand DNA generated by replication from a nearby replication origin (ARS306), we recently demonstrated that ribonucleotides are preferentially present in the nascent leading strand of the genome of a pol2-M644G rnh201Δstrain that is defective in RNase H2 (Lujan et al., 2012). As is displayed in Figure 1B, high mobility fragments are more abundant when RNase H2 is defective (Figure 1B, left panel, lane 3, pol2-M644G rnh201Δ) as compared to when it is not (lane 1, pol2-M644G RNH+). DNA fragments are even smaller upon deletion of both RNH201 and TOP1 (lane 4) as compared to the top1Δ or rnh201Δ mutant strains. When the experiment was performed in strains with the URA3 reporter placed in the opposite orientation, the same Top1-dependent difference in nascent leading strand fragment size was observed (Figure 1B, lanes 5 to 8). This difference was also observed when genomic DNA was subjected to enzymatic cleavage by RNase H2 (Figure S1). The gel images were scanned and quantified (Figure 1C/D and Supplemental Experimental Procedures), revealing a mean DNA fragment size of 940 bases in the pol2-M644G rnh201Δ mutant. This corresponds to approximately 13,000 (± 2500) ribonucleotides in the nascent leading strand of the 12 million base pair yeast genome. In comparison, the mean DNA fragment size in the pol2-M644G rnh201Δ top1Δ strain was 680 bases, corresponding to nearly 18,000 (±3900) ribonucleotides in the nascent strand. These data indicate that when RNase H2-dependent RER is defective in a pol2-M644G cells, approximately 5,000 ribonucleotides are removed from the genome by a Top1-dependent process.
Multistep DNA repair pathways must be carefully coordinated in order to avoid release of repair intermediates that can sometimes be more toxic to cells than the original lesion (Mol et al., 2000; Prasad et al., 2010; Williams et al., 2007; Wilson and Kunkel, 2000). We therefore hypothesized that Top1-dependent cleavage at ribonucleotides to generate unligatable DNA ends might underlie the adverse cellular consequences of ribonucleotides in the DNA of RNase H2-defective yeast. One such consequence is the activation of the S-phase checkpoint that was recently reported in a pol2-M644G rnh201Δ strain (Lazzaro et al., 2012). As a test of our hypothesis, we monitored checkpoint activation in this strain by immunoblotting for Rnr3, a subunit of ribonucleotide reductase (RNR) that is a sensitive readout of S-phase checkpoint activation (Davidson et al., 2012; Kumar et al., 2010). As expected, we observed an elevated level of Rnr3 protein in the pol2-M644G rnh201Δ mutant (Figure 2, lane 2). In contrast, no increase in Rnr3 level was detected in the pol2-M644G top1Δ strain (lane 3) or the pol2-M644G rnh201Δ top1Δ strain (lane 4). These results confirm that failure of RNase H2 to remove ribonucleotides incorporated by Pol ε triggers activation of the S-phase checkpoint in yeast. More importantly, they indicate that this activation is Top1-dependent, suggesting that the responsible lesions are dirty DNA ends resulting from Top1 incisions at ribonucleotides in DNA.
A pol2-M644G rnh201Δ strain has slightly elevated dNTP pools, strongly elevated rates of 2-5 base pair deletions in tandem repeat DNA sequences (Clark et al., 2011; Nick McElhinny et al., 2010a), and is sensitive to HU (Lazzaro et al., 2012), a drug that induces replication fork stalling by inhibiting dNTP synthesis. To determine if these cellular phenotypes depend on Top1, we compared the pol2-M644G rnh201Δ strain to a pol2-M644G rnh201Δ top1Δ strain lacking Top1-dependent ribonucleotide removal (Figure 3).
Flow cytometry analysis previously demonstrated that pol2-M644G and pol2-M644G rnh201Δ cells accumulate in S-phase and G2 (Nick McElhinny et al., 2010a). Although deletion of TOP1 does not affect cell cycle progression in a wild-type polymerase (POL2+) strain background, it does reduce the number of cells in G2 in a pol2-M644G rnh201Δ mutant (Figure 3A/B). These flow cytometry profiles reveal a more pronounced G1-peak and a less pronounced G2-peak in pol2-M644G rnh201Δ top1Δ cells. In accordance with the effect on cell cycle progression, deleting TOP1 in a pol2-M644G rnh201Δ mutant also reduces the total cellular dNTP level by 32% (Figure 3C), further indicating that replicative stress is reduced when Top1-nicking at ribonucleotides does not occur.
Next, we performed quantitative cell viability and spot dilution assays to determine survival in the presence of replication stress induced by HU. Consistent with previous results (Lazzaro et al., 2012), survival of a pol2-M644G rnh201Δ mutant is impaired in the presence of HU (Figure 3D), and the colonies that do grow are small and heterogeneous in size (Figure 3E). This HU-sensitivity was clearly displayed in the quantitative plating assay, where survival of the pol2-M644G rnh201Δ mutant was reduced by 30% compared to the control strains (Figure 3D and Figure S2). In both assays, deletion of TOP1 conferred a significant, albeit partial, reduction in HU sensitivity, indicating that Top1-dependent processing of unrepaired ribonucleotides contributes to replicative stress (Figure 3D/E).
A pol2-M644G rnh201Δmutant generates 2-5 base pair deletions at rates that are elevated by 100-fold or more compared to RNase H2-proficient strains (Nick McElhinny et al., 2010a). To determine if these signature mutations are Top1-dependent, we measured and compared the rates of mutations conferring 5FOA-resistance between a pol2-M644G rnh201Δ strain to a pol2-M644G rnh201Δ top1Δ strain (Table S1 and Figure S3). Sequencing the URA3 reporter enabled the use of this information to calculate the rate of 2-5 base pair deletions in tandem repeat sequences (Table S3). The results (Figure 3F and Figure S4) demonstrate that deleting TOP1 reduced the rate of 2-5 base pair deletions in tandem repeat sequences more than 300-fold. Deleting TOP1 in an rnh201Δ mutant encoding wild type Pol ε also strongly reduced the rate of 2-5 base pair deletions in URA3 (Figure 3F), as was observed for the CAN1 reporter gene (Kim et al., 2011).
RNase H2-dependent RER is very efficient at removing ribonucleotides incorporated into DNA during replication in vivo (Nick McElhinny et al., 2010a) and in vitro (Sparks et al., 2012). The present study indicates that there is a second ribonucleotide removal pathway initiated by Top1 cleavage at ribonucleotides, as detected here in rnh201Δ strains (Figure 1). This Top1-dependent process (Figure 4) has the capacity to remove a large number of ribonucleotides from DNA (Figure 1B/C and Figure S1). After Top1 incision, other currently unknown proteins are needed to excise the ribonucleotide and to process the 5′-OH and cyclic 2′-3′ phosphate ends of the DNA to allow ligation to complete repair (Figure 4, left).
Interestingly, Top1-dependent ribonucleotide removal is only apparent when RNase H2-initiated RER is defective (Figure 1B, lanes 3 and 7 versus lanes 4 and 8), not when it is active (lanes 2 and 6 versus lanes 1 and 5). Thus the major RNase H2-dependent RER pathway may exclude Top1 from cleaving ribonucleotides in DNA, and/or RNase H2 and Top1 may to some extent work on different subsets of ribonucleotides in the genome. These subsets could differ by nucleotide identity, configuration, local sequence context, chromosomal location or chromatin status. As Top1 plays a critical role in relieving supercoils that arise during transcription, it is also possible that the substrates for Top1-initiated repair may depend on transcriptional status. For example, it could be that R-loops formed during transcription are left unresolved when RNase H2 is defective, thereby creating substrates for Top1-mediated repair. Because a pol2-M644G rnh201Δ top1Δ strain is healthier than a pol2-M644G rnh201Δ strain (Figure 3), the main cellular benefit of Top1 cleavage at ribonucleotides may be related more to a role in transcription or some other process than to actually removing ribonucleotides from the genome. The detrimental effects of Top1-dependent ribonucleotide removal can then be likened to the known detrimental effects of another DNA repair process gone awry when processing a lesion, i.e., the cytotoxic effects of mismatch repair processing of alkylated bases (Cejka and Jiricny, 2008; Kat et al., 1993).
The results further suggest that Top1 is responsible for the replicative stress and genome instability (Figure 3) associated with ribonucleotides remaining in the nascent leading strand when RNase H2-dependent RER is defective, likely via incision at ribonucleotides in DNA by the endonuclease activity of Top1 (Figure 4). In the pol2-M644G rnh201Δ strain containing a substantial number of newly incorporated ribonucleotides that cannot be repaired by RNase H2, a correspondingly large number of dirty DNA ends may be generated. In a manner similar to the formation of toxic intermediates during base excision repair (Prasad et al., 2010; Wilson and Kunkel, 2000), a subset of these dirty DNA ends may escape completion of the reaction initiated by Top1-incision at a ribonucleotide, thereby eliciting replicative stress and activating the checkpoint response, e.g., by promoting replication fork stalling and/or collapse. This hypothesis invoking persistent dirty ends differs from the earlier proposal that the HU-sensitivity of the pol2-M644G rnh201Δ strain results from difficulty in replicating DNA containing unrepaired ribonucleotides (Lazzaro et al., 2012). The latter idea is somewhat disfavored by the observation here that, despite an increased density of unrepaired genomic ribonucleotides in nascent leading strand in pol2-M644G rnh201Δ top1Δ cells (Figure 1), these triple mutant cells do not have an activated S-phase checkpoint (Figure 2), nor are they sensitive to HU (Figure 3D/E). In such cells, pathways such as Rad5-dependent template switching and/or Pol ζ-dependent translesion DNA synthesis, which contribute to survival at least when both yeast RNases H are defective (Lazzaro et al., 2012), could also contribute to tolerating the more than 10,000 ribonucleotides present in the nascent leading strand (bottom right in Figure 4). In the future, it will be interesting to determine if Top1-dependent effects are also observed for ribonucleotides in the nascent lagging strand, whose enzymology for both replication (Kunkel, 2011) and repair (Hombauer et al., 2011; Liberti et al., 2012) differs significantly from that for the leading strand. Attempts are also underway to determine if Top1 initiates removal of ribonucleotides from the nuclear genome of cells with wild-type DNA polymerases, as suggested by the fact that deleting TOP1 in an rnh201Δ strain encoding wild type polymerases (POL+) strongly reduces the rate of 2-5 base pair deletions (Figure 3F and (Kim et al., 2011)).
RNase H2 and Top1 are highly conserved enzymes, suggesting that our present observations in yeast may be relevant to higher eukaryotes. For example, failure of RNase H2-dependent RER due to deletion of the mouse Rnaseh2b gene encoding a non-catalytic subunit of RNase H2 activates the p53-dependent DNA damage response and arrests cell proliferation in mouse embryos. The genomes of RNase H2-defective fibroblasts cultured from these embryos contain more than 1,000,000 ribonucleotides, and these cells are characterized by formation of micronuclei, large-scale cytogenetic anomalies and chromosomal rearrangements (Reijns et al., 2012). In humans, mutations in subunits of RNase H2 are associated with the rare autoinflammatory disorder, Aicardi-Goutières syndrome (AGS), a disease in which nucleic acids accumulate and trigger an antiviral immune response (Crow et al., 2006). Our data in yeast suggest that these types of genome instability may be initiated by Top1 incision at ribonucleotides that remain in the genome when RNase H2 is defective.
Genomic DNA was isolated from asynchronously growing cultures at mid-log phase (in YPDA at 30 °C) using the Epicentre Yeast DNA purification kit (MPY80200). Five μg of DNA was treated with 0.3 M KOH for 2 h at 55 °C, and subjected to alkaline hydrolysis and alkaline-agarose electrophoresis as described (Nick McElhinny et al., 2010a). The gel was neutralized and the DNA was transferred to a nylon membrane (Hybond N+) by capillary action in alkaline transfer buffer (0.4 N NaOH, 1 M NaCl) overnight. Southern analysis was performed using single-strand radiolabeled probes prepared from a PCR-amplified fragment of the URA3 reporter gene integrated at the AGP1 locus on chromosome III using a previously described procedure (Miyabe et al., 2011). Two opposite orientations of the URA3 reporter were used in this experiment to control for any potential differences in probe radiolabeling and/or hybridization. Depending on the orientation of the reporter gene, we can assign one of the two nascent DNA strands as leading (i.e. synthesized by Pol ε) based on replication fork movement from the nearest replication origin, ARS306. Using two orientations of the reporter gene allowed us to probe for alkali-sensitive sites in the nascent leading strand using two independent strand-specific probes (A or B). Quantitation of alkali-sensitive fragments was performed using ImageQuant software (GE Healthcare). The fraction was calculated by dividing the radioactive intensity (arbitrary units) at 0.1 mm intervals by the total intensity for each lane. The method of mean fragment size determination is described in the Supplementary Experimental Procedures.
Topoisomerase 1 initiates removal of ribonucleotides from yeast genomic DNA Ribonucleotides incorporated into nascent leading strand DNA are repaired by Top1 Top1 incision at ribonucleotides causes cellular stress and genome instability
We thank Michelle Heacock and Scott Williams for helpful comments on the manuscript and Kunkel lab members for data previously generated in the lab and useful discussions. We are grateful to Grace Kissling for statistical advice and Carl Bortner for expert assistance with flow cytometry analysis. We also acknowledge the National Institute of Environmental Health Sciences Molecular Genetics Core Facility for sequence analysis of 5-FOA-resistant mutants, and the Flow Cytometry Center for FACS analysis. 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), and by the Swedish Foundation for Strategic Research, the Swedish Research Council and the Swedish Cancer Society to A.C.
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