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. 2018 Apr 5;19(5):e45335. doi: 10.15252/embr.201745335

RNase H eliminates R‐loops that disrupt DNA replication but is nonessential for efficient DSB repair

Hongchang Zhao 1,†, Min Zhu 1,†, Oliver Limbo 1, Paul Russell 1,✉
PMCID: PMC5934767  PMID: 29622660

Abstract

In Saccharomyces cerevisiae, genome stability depends on RNases H1 and H2, which remove ribonucleotides from DNA and eliminate RNA–DNA hybrids (R‐loops). In Schizosaccharomyces pombe, RNase H enzymes were reported to process RNA–DNA hybrids produced at a double‐strand break (DSB) generated by I‐PpoI meganuclease. However, it is unclear if RNase H is generally required for efficient DSB repair in fission yeast, or whether it has other genome protection roles. Here, we show that S. pombe rnh1∆ rnh201∆ cells, which lack the RNase H enzymes, accumulate R‐loops and activate DNA damage checkpoints. Their viability requires critical DSB repair proteins and Mus81, which resolves DNA junctions formed during repair of broken replication forks. “Dirty” DSBs generated by ionizing radiation, as well as a “clean” DSB at a broken replication fork, are efficiently repaired in the absence of RNase H. RNA–DNA hybrids are not detected at a reparable DSB formed by fork collapse. We conclude that unprocessed R‐loops collapse replication forks in rnh1∆ rnh201∆ cells, but RNase H is not generally required for efficient DSB repair.

Keywords: double‐strand break repair, replication fork, R‐loop, RNase H1, RNase H2

Subject Categories: DNA Replication, Repair & Recombination

Introduction

Eukaryotic cells possess two types of RNase H enzymes, monomeric H1, and heterotrimeric H2, which are specifically tasked with eliminating RNA–DNA hybrids from the genome 1, 2. These hybrids arise by ribonucleotide incorporation into DNA during replication, or by formation of R‐loops during transcription, in which RNA anneals to the template DNA strand. All RNA–DNA hybrids potentially threaten genome stability, but R‐loops constitute a unique threat because they can potentially obstruct replisome progression, which can result in replication fork collapse or breakage 3, 4, 5, 6, 7, 8. These events have been most intently investigated in the budding yeast Saccharomyces cerevisiae, in which multiple studies have documented increased indicators of genome instability in strains lacking the RNase H enzymes 9, 10, 11, 12, 13, 14, 15, 16.

Homology‐directed repair (HDR) of DNA double‐strand breaks (DSBs) is essential for maintaining genome integrity 17, 18. In all HDR models, repair initiates with DNA end resection catalyzed by Mre11‐Rad50‐Nbs1 (MRN) protein complex and CtIP/Ctp1/Sae2, which interact to form a nuclease activity that generates a 3’ single‐strand DNA (ssDNA) overhang 19, 20. This ssDNA is rapidly coated with replication protein A (RPA), which is the major ssDNA‐binding protein in eukaryotic cells. Rad52 catalyzes replacement of RPA by Rad51 recombinase. The Rad51 filament performs the homology search and invasion of homologous DNA sequences in the intact sister chromatid or homologous chromosome, which is followed by DNA synthesis at the invading end 21. In mitotic cells, the repair of two‐ended DSBs, such as those formed by ionizing radiation (IR), is completed by a non‐crossover mechanism known as synthesis‐dependent strand annealing (SDSA). However, in the case of single‐ended DSBs formed by replication fork breakage, sister chromatid recombination (SCR) resets the replication fork in a mechanism that requires resolution of the recombination intermediate, either a D‐loop or Holliday junction 22.

Recently, a surprising requirement for RNase H in DSB repair was suggested in a study reporting that RNase H‐deficient cells of Schizosaccharomyces pombe are sensitive to expression of DNA endonucleases or exposure to several genotoxic agents 23. RNA–DNA hybrids accumulated around a DSB created by I‐PpoI homing endonuclease, and they increased further in RNase H‐defective cells. From these and other data, a model was proposed in which efficient DSB repair requires formation of RNA–DNA hybrids by RNA polymerase II transcription of ssDNA at resected DSBs, followed by RNase H‐mediated degradation of the RNA–DNA hybrids 23. Another recent study also linked RNase H to DSB repair, although in this case RNase H was proposed to specifically promote repair of DSBs caused by unprocessed R‐loops 24.

Given the importance of DSB repair, and the need to understand how defects in RNase H2 result in the crippling disease known as Aicardi‐Goutières syndrome 25, we set out to more fully assess the consequences of RNase H deficiency in fission yeast. Our findings show that RNase H is critical to eliminate RNA–DNA hybrids that trigger replication fork collapse, but RNase H is not generally required for efficient repair of DSBs.

Results

Increased markers of DNA damage in RNase H‐deficient cells

In S. cerevisiae, one consequence of RNase H deficiency is an increased number of Rad52 nuclear foci, which indicates increased DNA damage and correlates with an essential requirement for Rad52 in RNase H‐deficient cells 9, 24, 26. We examined Rad52 foci in fission yeast rnh1∆ cells lacking RNase H1, rnh201∆ cells lacking an essential subunit of RNase H2, or rnh1/201∆ cells lacking both RNase H enzymes. Whereas rnh1∆ or rnh201∆ single mutants were similar to wild type, each having about 2% of cells with Rad52 foci, this value increased to about 30% in rnh1/201∆ cells (Figs 1A and EV1).

Figure 1. Increased DNA damage in rnh1/201∆ cells.

Figure 1

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  1. Large increase in Rad52 foci in rnh1/201∆ cells. Rad52‐RFP was expressed from the endogenous locus. Error bars represent standard deviation of three independent biological experiments.
  2. Chk1‐dependent cell elongation in rnh1/201∆ cells. Box‐and‐whisker plots display the cell length of 100 septated cells. The bottom and top of the box are the first and third quartiles, respectively; the band inside the box is the median; the × is the mean; the ends of the whiskers represent the most extreme data still within 1.5 interquartile range (IQR); dots indicate data beyond 1.5 IQR. Statistical treatment: unpaired, two‐tailed Student's t‐test. ***P < 0.001.
  3. Increased Chk1 phosphorylation in rnh1/201∆ cells detected by immunoblotting. Controls include wild type exposed to IR (WT + IR).
  4. Poor growth of rnh1/201∆ cells, indicated by reduced colony size, is accentuated in chk1∆ and rad3∆ backgrounds. Fivefold serial dilutions of cells were incubated on YES plates at 32°C.

Figure EV1. Microscopic detection of Rad52‐RFP foci.

Figure EV1

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RNase H deficiency increases abundance of Rad52 foci. Scale bar is 20 μm.

An increased incidence of Rad52 foci is often associated with activation of the DNA damage checkpoint that delays the onset of mitosis. Consistent with this notion, rnh1/201∆ cells were significantly elongated at division (Fig 1B). This division delay substantially depended on Chk1 kinase (Fig 1B), which is essential for the DSB‐activated cell cycle checkpoint 27. However, the cell division delay of rnh1/201∆ cells was modest compared to ctp1∆ cells (Fig 1B), which are unable to repair most DSBs 28.

To further characterize rnh1/201∆ cells, we analyzed the Chk1 phosphorylation that is catalyzed by the master checkpoint kinase Rad3/ATR in response to DNA damage 29. Phosphorylated Chk1 was detected in untreated rnh1/201∆ cells (Fig 1C). These data indicate that rnh1/201∆ cells have increased DNA damage.

In spot dilution assays, rnh1/201∆ cells formed smaller colonies compared to wild type (Fig 1D), indicating an increased frequency of cell death. Culture doubling times were 2.49 h for wild type and 3.02 h for rnh1/201∆, calculated from cells grown in rich YES media at 30°C. The rnh1/201∆ growth defect was accentuated in the chk1∆ background, and further compromised in the absence of Rad3/ATR (Fig 1D), which activates both Chk1 and Cds1/Chk2 checkpoint kinases 30, 31.

From these results, we conclude that rnh1/201∆ cells accumulate DNA lesions that activate DNA damage checkpoints, which are critical for survival in the absence of RNase H.

RNase H‐deficient cells are largely resistant to replication fork collapse caused by camptothecin

Fission yeast rnh1/201∆ cells are sensitive to several genotoxins that cause replication stress, including the topoisomerase I (Top1) inhibitor camptothecin (CPT) and the ribonucleotide reductase inhibitor hydroxyurea (HU) 23. CPT stabilizes Top1 cleavage complexes (Top1ccs) at single‐strand DNA breaks (SSBs), which collapse replication forks in S‐phase. HU lowers the effective concentration of dNTPs, thereby slowing or stalling DNA replication forks, which can eventually cause replication fork collapse. To better understand the genotoxin sensitivities of rnh1/201∆ cells, we compared them directly to rad50∆ cells that are unable to repair collapsed forks. Whereas rad50∆ cells were acutely sensitive to 0.25 μM CPT in plating assays, comparable sensitivity of rnh1/201∆ cells only emerged at 20‐ to 40‐fold higher amounts of CPT (Fig 2A). We confirmed these differences in short‐term exposure assays, in which rad50∆ cells steadily lost viability during a 4‐h exposure to 20 μM CPT, whereas rnh1/201∆ cells maintained high viability (Fig 2B).

Figure 2. RNase H‐deficient cells are weakly sensitive to camptothecin.

Figure 2

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  1. Plating assays indicate rnh1/201∆ cells are weakly sensitive to CPT. Fivefold serial dilutions of cells were incubated on plates containing the indicated concentrations of CPT. Plates were photographed after 3‐day incubation at 32°C. Note rad50∆ and rnh1/201∆ cells form smaller colonies, indicating increased cell death.
  2. In transient exposure assays, rnh1/201∆ cells are only weakly sensitive to CPT. Cells were exposed to 20 μM of CPT for 0–4 h. Bars represent standard deviation of three independent biological experiments.
  3. Plating assays indicate rnh1/201∆ cells are moderately sensitive to HU.
  4. In transient exposure assays, rnh1/201∆ cells are moderately sensitive to HU. Cells were treated with the indicated doses of HU for 6 h. Bars represent standard deviation of three independent biological experiments.

A different picture emerged in HU sensitivity assays, in which rad50∆ and rnh1/201∆ cells were nearly equally sensitive in spot dilution assays (Fig 2C). The rnh1/201∆ cells were actually more sensitive to HU in acute exposure assays (Fig 2D). Interestingly, rnh201∆ cells displayed some sensitivity to HU, whereas rnh1∆ cells were fully resistant (Fig 2C). These findings correlate with the ability of RNase H2, but not RNase H1, to process single ribonucleotides incorporated into DNA 1.

From these data, we conclude that at least the large majority of CPT‐induced collapsed replication forks are repaired in rnh1/201∆ cells. The stronger HU sensitivity of rnh1/201∆ cells, as compared to a rad50∆ mutant that is acutely defective in DSB repair, suggests that RNase H likely has important functions in HU resistance that are not required for DSB repair.

DSBs created by ionizing radiation are efficiently repaired without RNase H

To more directly explore the role of RNase H in DSB repair, we performed survival assays using IR emitted from a cesium‐137 source 32, 33. Whereas rad50∆ cells displayed extreme IR sensitivity in spot dilution assays, neither rnh1∆ or rnh201∆ single mutants, nor the rnh1/201∆ double mutant, displayed any increased sensitivity to IR (Fig 3A). To confirm and quantify these findings, we repeated the experiment and scored colony formation. Normalizing values to mock‐irradiated cells, we again found that rad50∆ cells were acutely sensitive to IR, whereas the rnh1/201∆ strain did not differ significantly from wild type (Fig 3B). We conclude that RNase H is not required to efficiently repair IR‐induced DSBs.

Figure 3. RNase H‐deficient cells are insensitive to ionizing radiation.

Figure 3

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  1. RNase H is not required for IR survival. Cells exposed to IR from a cesium‐137 source were plated with fivefold serial dilutions. Plates were photographed after 3‐day incubation at 32°C.
  2. Quantitative analysis confirms that rnh1/201∆ cells are insensitive to IR. Bars represent standard deviation of three independent biological experiments.

Efficient SDSA and SCR repair of the mat1 DSB in rnh1/201∆ cells

Having found that RNase H‐deficient cells are insensitive to IR, we next investigated repair of the site‐specific DSB that triggers switching between plus and minus mating types in homothallic fission yeast 34, 35, 36. This DSB, which occurs every other cell cycle, is formed by replication fork collapse at a SSB near the expressed mat1 locus (Fig 4A). Repair occurs by intrachromosomal SDSA using transcriptionally silent mat2 or mat3 donor templates. Key HDR proteins (e.g., Rad50, Rad51, and Rad52) are critical for growth in mating type switching‐proficient h 90 backgrounds 22, 37. In contrast, we observed no enhanced growth defects of rnh1/201∆ cells in the h 90 background (Fig 4B). Mating type switching competency was assessed by exposing cells to iodine vapor, which stains starch produced in spores formed after mating 38. The iodine staining of h 90 rnh1/201∆ colonies was nearly as strong as h 90 wild type, indicating efficient mating type switching in the rnh1/201∆ cells (Fig 4B).

Figure 4. DSB repair at the mat1 broken replication fork occurs efficiently in the absence of RNase H.

Figure 4

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  1. Mating type switching system in fission yeast. See text for details. Lower panel shows repair mechanism in mat2,3∆ donorless strain.
  2. Proficient mating type switching in h 90 rnh1/201∆ cells. Colonies of the indicated genotypes on SSA plates were exposed to iodine vapor to assess mating/sporulation efficiency. Controls include wild‐type h 90, non‐switchable h −, and switching‐defective swi3∆ h 90.
  3. Rad50 is required SCR repair of the DSB at the mat1 locus in mat2,3∆ cells. The rad50∆ mat2,3∆ cells display very poor growth compared to single mutants or wild type. Products of a tetrad dissection were photographed on successive days.
  4. RNase H is not required for SCR repair of the DSB at the mat1 locus in mat2,3∆ cells. The rnh1/201∆ mat2,3∆ cells display no growth defect relative to rnh1∆ rnh201∆.

We have previously described a mat2,3∆ “donorless” strain that lacks the heterochromatic silent mating type cassettes 22, 39. Deprived of the intrachromosomal SDSA repair templates, these cells instead use interchromosomal sister chromatid recombination (SCR) to repair the mat1 DSB (Fig 4A). This system is a model for replication fork breakage caused by genotoxins or genetic defects that create SSBs 22, 40. We used the mat2,3∆ system to investigate whether RNase H is required for SCR. Whereas rad50∆ mat2,3∆ colonies displayed acute growth defects as expected, we observed no enhanced growth defects in the rnh1/201∆ mat2,3∆ background (Fig 4C and D).

From these data, we conclude that RNase H is not required to efficiently repair the mat1 DSB by intrachromosomal SDSA or interchromosomal SCR.

RNA–DNA hybrids are not enriched at the reparable mat1 DSB in rnh1/201∆ cells

Abundant RNA–DNA hybrids were detected near a DSB in chromosome II generated by I‐PpoI meganuclease 23. To investigate whether this is a general phenomenon, we assessed whether RNA–DNA hybrids are formed at the mat1 DSB. A key experimental difference is that endonucleases can cleave both sister chromatids, which eliminates the repair template, whereas fork breakage leaves an intact sister chromatid sequences opposite the DSB. Control chromatin immunoprecipitation (ChIP) experiments confirmed that Rad52 strongly enriches DNA sequences adjacent to the mat1 DSB (BE primer set) and past the break site (SC primer set), as compared to a site 10 kb away (Fig 5A and B). The Rad52 SC enrichment is expected from strand invasion into the sister chromatid during SCR. We next used the S9.6 antibody, which binds RNA–DNA hybrids, in a DNA–RNA immunoprecipitation (DRIP) protocol 41, 42. In mat2,3∆ cells, we detected a weak BE signal (~0.01% of input) that was sensitive to RNase H treatment in vitro, but the signal was nearly the same in the rnh1/201∆ background, and it was approximately equivalent to the SC signal and the 10 kb control site (Fig 5C). To validate the assay, we analyzed sequences at a single tRNATyr gene (SPBTRNATYR.03), as S. cerevisiae studies showed that RNase H processes RNA–DNA hybrids at tRNA genes 43, 44, 45. DRIP detected an ~10‐fold higher enrichment of RNA–DNA at this tRNA gene in rnh1/201∆ cells relative to the wild‐type control (Fig 5C). From these data, we conclude that RNase H eliminates R‐loops formed at the tRNATyr gene, but RNA–DNA hybrids fail to accumulate at the mat1 DSB in the presence or absence of RNase H. The latter result is consistent with the efficient growth of rnh1/201∆ cells in h 90 and mat2,3∆ backgrounds.

Figure 5. RNA–DNA hybrids are not enriched at the reparable mat1 DSB.

Figure 5

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  1. Diagram of the mat2,3∆ broken replication fork showing the location of PCR products used for ChIP and DRIP assays.
  2. Rad52 is enriched at the mat1 DSB site and the sister chromatid region used for HDR. ChIP assay was performed with Rad52‐5FLAG expressed from the endogenous locus in a mat2,3∆ strain. Relative enrichment was calculated as the percentage of ChIP/input and presented as the mean of three technical replicates. The results were replicated in three independent experiments.
  3. RNA–DNA hybrids are enriched at the tRNATyr gene, but not at the reparable DSB at the mat1 locus in rnh1/201∆ cells. DRIP assay was performed with S9.6 antibody using the indicated strains with or without RNase H treatment. Relative enrichment was calculated as ChIP/input. Relative enrichment was calculated as the percentage of ChIP/input and presented as the mean of three technical replicates. The results were replicated in three independent experiments.

Rad50, Ctp1, and Mus81 are essential in rnh1/201∆ cells

The increased Rad52 foci, Chk1 phosphorylation, and requirement for Rad3/ATR in rnh1/201∆ cells (Fig 1) indicate that persistent R‐loops cause DSBs in fission yeast. In support of this conclusion, tetrad analyses revealed that rad50∆ rnh1/201∆ cells are inviable (Fig 6A). To confirm this finding, we analyzed the requirement for Ctp1 in the absence of RNase H. We observed that ctp1∆ rnh1/201∆ spores also failed to generate viable cells (Fig 6B).

Figure 6. HDR‐mediated reset of collapsed replication forks is essential in RNase H‐deficient cells.

Figure 6

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  1. Tetrad analysis showing that Rad50 is essential in rnh1/201∆ background.
  2. Tetrad analysis showing that Ctp1 is essential in rnh1/201∆ background.
  3. Tetrad analysis showing that Mus81 is essential in rnh1/201∆ background.

The MRN protein complex, as well as Ctp1, Rad51, and Rad52, is required for SDSA repair of two‐ended DSBs formed by ionizing radiation and SCR repair of one‐ended DSBs formed by replication fork collapse. However, in mitotic cells, the Mus81‐Eme1 resolvase is only required for SCR repair of collapsed forks (Fig EV2A and B). These conclusions comport with the acute CPT sensitivity of mus81∆ cells, and there inviability in the mat2,3∆ background, as well as their full IR resistance and mating type switching proficiency 22, 46, 47, 48, 49, 50. Tetrad analysis revealed that Mus81 is essential in rnh1/201∆ cells (Fig 6C). From these results, we conclude that rnh1/201∆ cells suffer increased rates of replication fork collapse, forming DSBs that are repaired by SCR requiring MRN‐Ctp1 and Mus81, but not RNase H.

Figure EV2. DSB repair mechanisms.

Figure EV2

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  1. Repair of a broken replication fork. Replication fork breaks when it encounters a SSB in the leading strand template. The intact sister serves as a template for homology‐directed repair of the resected single‐ended DSB. Restoration of the replication fork creates an inter‐sister joint DNA molecule, a D‐loop or Holliday junction, that must be resolved by Mus81‐Eme1 to allow chromosome segregation during mitosis.
  2. Repair of IR‐induced DSBs. DSBs created by IR in G2 phase, which accounts for most of the cell cycle in fission yeast, are repaired by SDSA. The intact sister serves as a template for homology‐directed repair of a resected DNA end. The invading DNA strand is extended by DNA synthesis, after which it is displaced from the sister chromatid, allowing it to anneal to the complementary DNA strand across the DSB. SDSA does not require resolution of joint DNA molecules by Mus81‐Eme1.
  3. Processing of DSBs created by site‐specific endonucleases. Homing endonucleases and restriction endonucleases are able to cut both sister chromatids, which eliminates SDSA as a repair mechanism. The DSBs become hyper‐resected.

Discussion

In this study, we have analyzed the roles of RNases H1 and H2 in preserving genome integrity in fission yeast. From these experiments, we draw two major conclusions: (i) rnh1/201∆ cells suffer increased DNA damage from collapsed replication forks; (ii) efficient DSB repair does not generally require RNase H.

Regarding DSBs, we analyzed physiologically relevant situations of DSB formation and repair. Ionizing radiation is ideally suited for this purpose, as it rapidly and directly creates DSBs randomly throughout the genome, and it does so in a strict dose‐dependent manner. IR sensitivity is a defining property of HDR mutants. Indeed, many of the key HDR genes, including RAD50, RAD51, and RAD52, were discovered in screens of IR‐sensitive mutants 51. We found that rnh1/201∆ cells are completely insensitive to IR. We obtained this result in multiple independent experiments using rigorously characterized strains. We also found that rnh1/201∆ cells are only weakly sensitive to CPT and are fully able to repair the mat1 DSB. We conclude that RNase H is not generally required to repair DSBs in fission yeast.

As each of the ~150 ribosomal DNA (rDNA) genes arrayed at the ends of chromosome III has a I‐PpoI site 52, I‐PpoI expression might constitute a unique situation in which RNase H promotes DSB repair 23. Due to its repetitive structure and high rate of transcription, the rDNA locus is susceptible to genetic instability and subject to additional regulation of homologous recombination 53. Notably, Top1‐dependent removal of DNA torsional stress is critical for stability of the rDNA locus, which explains rDNA genetic instability in CPT‐treated cells 54. RNase H prevents R‐loop accumulation in the rDNA of S. pombe and S. cerevisiae (Fig EV3A) 42, and rnh1/201∆ cells display increased markers of DNA damage in the rDNA 15, 24, 55. Replication fork collapse triggered by unprocessed R‐loops in the rDNA is likely a key reason why HDR is essential in rnh1/201∆ cells. Synergism of these events with abundant cleavage of the rDNA could account for the I‐PpoI sensitivity of rnh1/201∆ cells. We attempted to investigate this possibility by analyzing strains that lack the natural I‐PpoI sites in the rDNA (rDNA I‐PpoImt), but have a I‐PpoI site engineered at the lys1 locus in chromosome II 52. However, transient I‐PpoI expression caused acute lethality in all rDNA I‐PpoImt backgrounds, including wild type, rnh1/201∆, and ctp1∆ (Fig EV3B and C). These results are expected if I‐PpoI efficiently cuts both sister chromatids in the majority of cells, which destroys the template used for HDR (Fig EV2C) 52, 56, 57, 58, 59.

Figure EV3. Analysis of rDNA and I‐PpoI site.

Figure EV3

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  1. RNA–DNA hybrids are enriched in the rDNA but not at the reparable DSB at the mat1 locus in rnh1/201∆ cells. DRIP assay was performed with S9.6 antibody or no antibody control (−Ab) using the indicated strains. RNA–DNA hybrids were enriched in the 5′ region of the 18s rRNA in the rDNA using two different primer pairs (18S‐5′‐1 and 18S‐5′‐2). Relative enrichment was calculated as the percentage of ChIP/input and presented as the mean of three technical replicates. The results were replicated in three independent experiments.
  2. RNase H does not impact I‐PpoI sensitivity in strains with a single I‐PpoI site. Strains derived from YSLS792 52, which lacks the natural I‐PpoI site in the rDNA but has a single I‐PpoI site engineered in chromosome II, and expresses I‐PpoI from an ahTET‐inducible promoter, were grown in the presence of ahTET for 1.5 h prior to plating on YES media. Controls include cells grown in the absence of ahTET and plated on YES media (no treatment) or the indicated concentrations of CPT or HU. Plates were photographed after 3‐day incubation at 32°C.
  3. I‐PpoI site cutting efficiency for experiment shown in panel (B). Genomic DNA extracted from cells grown in the absence (−No) or presence (−ahTET) of ahTET for 1.5 h was amplified with the primers as indicated. CS primer: flanking the I‐Ppol site engineered at the lys1 locus. HIS(NTC): non‐targeted control primer. rDNA CS Primer: Primer which are flanking the I‐Ppol cutting site at the rDNA locus.
  4. RNA–DNA hybrid enrichment around the I‐Ppol‐induced DSB site at the lys1 locus (lys1::I‐PpoI CS ‐hph) in the rDNA I‐PpoImt strain that lacks the I‐PpoI site in the rDNA repeats. DRIP assay was performed with S9.6 antibody using genome DNA extracted from cells with or without ahTET treatment. Primers amplified sequences 0.5 or 1.0 kb from the DSB, or at a control site at the his3 locus [HIS(NTC)]. Relative enrichment was calculated as the percentage of ChIP/input and presented as the mean of three technical replicates. The results were replicated in three independent experiments.
  5. I‐PpoI cutting efficiency for experiment shown in panel (D). CS Primer: flanking the I‐Ppol site at the lys1 locus. rDNA CS Primer: primer flanking the I‐Ppol site at the rDNA locus, which is mutated to be I‐Ppol resistant. HIS(NTC): non‐targeted control primer.

We observed no evidence for the accumulation of RNA–DNA hybrids at the mat1 DSB, either in wild‐type or rnh1/201∆ cells. This result contrasts with the large accumulation of RNA–DNA hybrids detected around a cleaved I‐PpoI site engineered in chromosome II, which increased another ~2‐fold in rnh1/201∆ cells 23. We considered whether the massive cleavage of the rDNA by I‐PpoI might be connected to the appearance of RNA–DNA hybrids around the I‐PpoI DSB site in chromosome II, but we observed a large increase in RNA–DNA hybrids flanking the cleaved I‐PpoI site at the lys1 locus in the rDNA I‐PpoImt strain lacks that the I‐PpoI site in the rDNA repeats (Fig EV3D and E). Additional studies will be required to address this issue, but we note that the circumstances of DSB formation and repair are quite different in the two systems. Efficient cutting by sequence‐specific endonucleases results in both sister chromatids suffering DSBs at precisely the same site (Fig EV2C), which negates HDR as a repair mechanism unless there is an ectopic repair template that lacks the endonuclease cleavage site 56, 57. Depending on the experimental parameters, a culture may have some cells that have cleaved both sister chromatids, while others that have cleaved only one sister. The irreparable DSBs in the former class will become hyper‐resected, typically having 10 kb or more of ssDNA overhangs 58, 60. These aberrant overhangs might become substrates for the RNA transcription machinery, which could explain why RNA–DNA hybrids are detected at I‐PpoI DSB sites in chromosome II.

RNase H‐deficient cells were also reported to be sensitive to expression of SmaI endonuclease, which in principle creates many paired blunt‐end DSBs in sister chromatids of the fission yeast genome 23. Mutants lacking critical HDR proteins were not tested in these investigations. Studies in budding yeast showed that blunt DNA ends are poor substrates for HDR 61. Indeed, wild‐type, rad50∆, and rad52∆ strains were killed at equal rates by expression of a blunt‐end cutting endonuclease in S. cerevisiae 61. It is unclear if this is a general phenomenon.

The mild CPT sensitivity of S. pombe rnh1/201∆ cells, which mirrors S. cerevisiae studies 26, indicates that RNase H is not generally required to repair DSBs at collapsed replication forks. This conclusion is supported by the efficient repair of the mat1 DSB. In S. cerevisiae, the modest CPT sensitivity of rnh1/201∆ cells was attributed to R‐loop accumulation in the rDNA, because genetic elimination of Top1 increases rDNA R‐loops 26, 42. HU treatment increases ribonucleotide incorporation into DNA, which are primarily excised by RNase H2‐initiated ribonucleotide excision repair (RER) 1, 62. This mechanism likely explains why rnh201∆ cells are modestly sensitive to HU. As previously proposed 9, 26, it is therefore likely that increased ribonucleotide incorporation into DNA, coupled with defects in RER, R‐loop processing, and RNA primer removal, underlies the HU sensitivity of rnh1/201∆ cells.

We found that rnh1/201∆ cells experience increased DNA damage, as shown by Rad52 foci, Chk1 phosphorylation, and poor growth in chk1∆ or rad3∆ backgrounds (Fig 1). Rad50 and Ctp1 are essential in rnh1/201∆ cells, and thus, this DNA damage likely involves DSBs. The requirement for Mus81 in rnh1/201∆ cells is most straightforwardly explained by increased replication fork collapse, as Mus81 is essential to complete SCR repair of DSBs at broken replication forks, while it plays no role in SDSA repair of IR‐induced DSBs (Figs 7 and EV2) 22, 46, 47, 48, 49, 50. The increased R‐loops in rnh1/201∆ cells (Figs 4C and EV3A) likely cause replication fork collapse, which is consistent with S. cerevisiae studies 10. Lingering RNA primers from Okazaki fragments might also be involved 14. In some circumstances, R‐loops might trigger DSBs independently of replication 24, but repair of these DSBs should not require Mus81 unless this repair occurs by break‐induced replication (BIR) 63. No matter how R‐loops cause DSBs, it is reasonable to propose that their persistence at DSBs in rnh1/201∆ cells might interfere with repair 24, making this repair less efficient than at DSBs formed by IR or other clastogens. This possibility is consistent with the reduced growth rate of rnh1/201∆ cells. However, this idea is completely different from the concept that synthesis and degradation of RNA–DNA hybrids is centrally involved in the basic mechanism of homology‐directed repair.

Figure 7. RNases H1/H2 eliminate R‐loops and RNA–DNA hybrids to allow efficient DNA replication.

Figure 7

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In the absence of RNases H1 and H2, replication forks collapse at unprocessed R‐loops and RNA–DNA hybrids. These collapsed forks are repaired by sister chromatid recombination (SCR), which creates inter‐sister junctions that are resolved by Mus81‐Eme1 resolvase. Efficient repair of DSBs does not generally require RNases H1/H2.

In summary, our data provide compelling evidence that RNase H is needed to eliminate RNA–DNA hybrids that trigger replication fork collapse, but it is not generally required for efficient DSB repair.

Materials and Methods

Strain construction and analysis

Standard methods were used for constructing and culturing strains 38. Unless otherwise noted, YES (yeast extract, glucose, and supplements) or versions of EMM (Edinburgh minimal media) were used for all experiments. Tetrads were photographed after 3–5 days at 32°C. All strains are listed Table EV1.

I‐PpoI cutting assay

Cells were cultured to log‐phase in EMMG at 30°C, at which point ahTET (Sigma‐Aldrich 37919) was added to a concentration of 2.5 μg/ml to induce I‐Ppol expression for 1.5 h. For plating assays, cells were washed three times in EMMG without ahTET before dilution on YES plates.

DNA damage sensitivity assays

Cells were grown in liquid media to log‐phase, adjusted to OD600 = 0.4, then spotted onto plates using fivefold serial dilutions. For ionizing radiation, cells were exposed to a 137Cs source or mock‐treated. Alternatively, cells were plated on media containing the indicated concentrations of genotoxins. Plates were incubated at 32°C for 3 days before imaging. For quantitative survival assays, log‐phase cells were treated with HU for 6 h at the indicated dosage, or exposed to 20 μM CPT for the indicated duration, or exposed to the 137Cs source, or mock‐treated, and then plated at a density of 300 cells/plate. After 4‐day incubation at 32°C, colonies were counted and normalized to mock‐treated values. Standard deviations were determined from minimum of three assays.

ChIP and DRIP

For ChIP, log‐phase cells grown in YES liquid media 100‐ml cultures at 32°C were cross‐linked in 1% formaldehyde for 15 min at room temperature, followed by addition of 5‐ml 2.5 M glycine for 5 min, harvested by centrifugation, washed three times in cold water, and flash‐frozen. 50 OD600 cells were suspended in 600 μl low triton lysis buffer (50 mM HEPES/KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 0.1% TX‐100, 0.1% Na‐deoxycholate) and then disrupted by glass bead beating in a FastPrep‐24 (MP Biomedical) homogenizer. After a short centrifugation, the pellet was suspended with high triton lysis buffer (50 mM HEPES/KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% TX‐100, 0.1% Na‐deoxycholate) with Yeast Protease Inhibitor Cocktail (Sigma‐Aldrich). Chromatin was sheared with a Bioruptor (Diagenode) ultrasonicator to the size range around 500 bp. Samples were centrifuged and supernatant collected, retaining 50 μl of lysate as whole‐cell DNA input. Antibody‐coupled Dynabeads Protein G (Thermo Fisher) were used for immunoprecipitations performed overnight at 4°C. Anti‐FLAG M2 (Sigma‐Aldrich) was used to immunoprecipitate FLAG‐tagged Rad52. Uncoupled Dynabeads were used as minus‐antibody (−Ab) control. Beads were washed once in each of the following buffers for 5 min at 4°C: wash buffer I (50 mM HEPES/KOH, pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% TX‐100, 0.1% Na‐deoxycholate), wash buffer II (50 mM HEPES/KOH, pH 7.5, 500 mM NaCl, 1 mM EDTA, 1% TX‐100, 0.1% Na‐deoxycholate), wash buffer III (10 mM Tris, pH 8.0, 0.25 M LiCl, 0.5% NP‐40, 0.5% Na‐deoxycholate, 1 mM EDTA), and wash buffer IV (50 mM Tris, pH 8.0, 10 mM EDTA). Washed beads were suspended in 0.1 ml 10% Chelex‐100 (Bio‐Rad), vortexed, boiled for 10 min, and cooled to room temperature. This sample was added to 5 μl of 20 mg/ml Proteinase K (Thermo Fisher), followed by incubation at 55°C for 30 min while shaking. Beads were boiled for another 10 min. Sample was centrifuged and supernatant collected. Beads were suspended with 100 μl 2× TE to the beads, vortexed, centrifuged, and supernatants pooled. qPCR analysis was performed with iTaq Universal SYBR Green Supermix (Bio‐Rad) on an CFX Connect™ Real‐Time PCR Detection System (Bio‐Rad) using Taq DNA polymerase (Sigma‐Aldrich). Data were analyzed using the comparative CT method 64. Fold enrichment was calculated as ChIP/Input. Primers used for qPCR are listed in Table EV2.

For the DRIP experiment shown in Fig EV3A, the ChIP protocol was used with the following modifications: (i) no formaldehyde cross‐linking; (ii) the RNA–DNA hybrids were precipitated with antibody S9.6 (Kerafast) 41. For all other DRIP experiments, total nucleic acids were extracted by glass bead beating in phenol:chloroform. The S9.6 antibody (2 μg) was coupled with 100 μl of protein G magnetic beads (Invitrogen). DNA was recovered with Chelex‐100. qPCR analysis was performed with enrichment calculated by ratio of DRIP/input. For experiments with the RNase H treatment control, washed beads were re‐suspended in 300 μl of RNase H reaction buffer containing (NEB, M0297L) containing 4% glycerol and 20 mg/ml BSA. Beads were incubated for 2.0 h at 37°C in the absence or presence of 15 μl of recombinant Escherichia coli RNase HI (75 units, NEB, M0297L), with shaking at 1,000 rpm (Eppendorf Thermomixer). Reactions were stopped by adding 10 mM EDTA. Beads were washed successively with buffers as above, and DNA was recovered with Chelex‐100 and analyzed by qPCR.

Live cell microscopy

Cells were photographed using a Nikon Eclipse E800 microscope equipped with an INFINITY3‐1 charge‐coupled device (CCD) camera and INFINITY ANALYZE software (Lumenera). Rad52‐red fluorescence protein (RFP) expressing strains were grown in EMM (Edinburgh Minimal Media) until mid‐log‐phase for focus quantification assays. Quantification was performed by scoring 1,000 or more nuclei from three independent experiments.

Immunoblots

Exponential phase cells grown in YES were exposed to 90 Gy of IR using a 137Cs source or left untreated before harvesting. For the Chk1 mobility shift assay, whole‐cell extracts were prepared from exponentially growing cells in standard lysis buffer. Protein amounting to 150 μg was resolved by SDS–PAGE using 15‐by‐18‐cm 8% gels with an acrylamide/bisacrylamide ratio of 99:1. Proteins were transferred to nitrocellulose membranes, blocked with 5% milk in TBST (0.05% Tween), and probed with anti‐HA (12CA5) antibody (Roche catalog no. 11583816007). Ponceau‐stained blots were used for a loading control. The data shown are representative of at least two independent experiments.

Cell length measurements

Log‐phase cells grown in YES at 32°C were photographed using a Nikon Eclipse E800 microscope equipped with an INFINITY3‐1 CCD camera. The length of at least 100 septating cells was measured using ImageJ software.

Author contributions

Conceptualization: HZ, PR; funding acquisition: PR; investigation: HZ, MZ, OL; writing: HZ, PR.

Conflict of interest

The authors declare that they have no conflict of interest.

Supporting information

Expanded View Figures PDF

Click here for additional data file. (725KB, pdf)

Table EV1

Click here for additional data file. (38.5KB, doc)

Table EV2

Click here for additional data file. (17.4KB, docx)

Review Process File

Click here for additional data file. (325.9KB, pdf)

Acknowledgements

We thank Nick Boddy for helpful discussions. This work was supported by NIH grants GM059447, CA077325, and CA117638.

EMBO Reports (2018) 19: e45335

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Expanded View Figures PDF

Click here for additional data file. (725KB, pdf)

Table EV1

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Table EV2

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Review Process File

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