Sae2/CtIP prevents R-loop accumulation in eukaryotic cells
- Howard Hughes Medical Institute, The University of Texas at Austin, United states
- The University of Texas at Austin, United States
- National Cancer Institute, National Institutes of Health, United States
- University of Arkansas for Medical Sciences, United States
Figures
Figure 1 with 2 supplements
Transcription termination factors suppress DNA damage sensitivity of sae2Δ and mre11 nuclease-deficient strains.
(A) Full-length PCF11, SSU72, RTT103, SEN1, and sen1 mutants G1747D and R302W were expressed from a 2μ plasmid in sae2Δ cells. Fivefold serial dilutions of cells expressing the indicated Sae2 …
Figure 1—figure supplement 1
Overexpression of SSU72, NRD1, RTT103, or YSH1 does not complement Δsae2 strains for DNA damage sensitivity.
Overexpressed genes were expressed from a 2μ plasmid. Fivefold serial dilutions of yeast strains were plated on nonselective media (untreated) or media containing camptothecin or MMS and grown for …
Figure 1—figure supplement 2
SEN1 overexpression does not complement the resection deficiency in Δsae2 yeast strains.
Wild-type, Δsae2, Δsae2 + wild type SAE2, and Δsae2 + SEN1 strains containing a galactose-inducible HO endonuclease and an HO cut site in a LEU2 cassette separated from a homologous LEU2 cassette 25 …
Figure 2
Sae2 associates with sites of high levels of transcription which accumulate R-loops in the absence of Sae2.
(A) The survival of wild-type and sae2Δ strains was measured by exposing cells to camptothecin (100 μM for 2 hr) while in G1 phase or S phase and plating cells on rich media. The percentage of …
Figure 3
RNA Polymerase stalling at highly transcribed genes is exacerbated in sae2Δ strains with DNA damage.
(A) HTB-tagged Rpb2 (a component of RNA Pol II) levels were measured at various genes during S phase in wild-type and sae2Δ strains with or without CPT exposure and in the presence or absence of …
Figure 4 with 2 supplements
CtIP deficiency induces R-loop accumulation at sites of DNA damage.
(A) CtIP-depleted U2OS cells complemented with vector only or constructs overexpressing eGFP-CtIP, PCF11, or Senataxin (C-terminus) as indicated were exposed to increasing concentrations of CPT (1 …
Figure 4—figure supplement 1
Senataxin overexpression partially rescues growth but not resection in CtIP-depleted human cells.
(A) CtIP depletion and the expression of eGFP-tagged wild-type CtIP was monitored by western blot with antibody directed against CtIP (Active Motif, #61141). (B) CtIP depletion with shRNA was …
Figure 4—figure supplement 2
There is no effect of CtIP depletion on mCherry independent of RNaseH.
(A) Imaging of RNaseHD10R-E48R-mCherry or mCherry only was performed in U2OS cells with CtIP depletion as indicated. 7 and 3 cells were imaged for RNaseHD10R-E48R-mCherry and mCherry-only …
Figure 5 with 1 supplement
CtIP depletion affects R-loop accumulation and transcription in human cells.
(A) DNA-RNA hybrids were quantified in undamaged U2OS cells by monitoring chromatin-bound RNaseHD10R-E48R-mCherry by FACS. 10,000 cells were counted in each of 3 biological replicates; error bars …
Figure 5—figure supplement 1
CtIP-depleted human cells show alterations in transcription of endogenous genes.
(A) (B) Quantification of mRNA expression by RT-qPCR in U2OS cells with or without CtIP shRNA as indicated, measuring transcripts at the UBF and CD30 genes. (C) Quantification of mRNA expression in …
Figure 6 with 3 supplements
CtIP depletion induces accumulation of R-loops in human cells.
(A) RNA/DNA hybrids were quantified by DRIP-qPCR in U2OS cells containing a stably integrated, doxycycline-inducible transgene containing murine Sγ3 repeats. R-loop accumulation was measured by …
Figure 6—figure supplement 1
Sγ3 transcript levels are similar in wild-type and CtIP-depleted cells.
(A) Quantification of mRNA expression by RT-qPCR. A dox-inducible construct with murine Sγ3 repeats was stably integrated in U2OS cells, and the levels of the Sγ3 mRNA transcripts were measured in …
Figure 6—figure supplement 2
CtIP depletion generates R-loops at the CD30 and UBF loci in human cells.
Levels of RNA-DNA hybrids were measured with DRIP-qPCR as in Figure 6 at the CD30 (A) and the UBF (B) loci; n > 3, error bars represent S. D. * denotes p < 0.05 using Student's two-tailed T test …
Figure 6—figure supplement 3
DRIP-qPCR signal is eliminated by RNaseH treatment in vitro.
(A) Diagram of qPCR primer locations in the RPL13A gene. DRIP-qPCR was performed in wild-type and CtIP-depleted cells as in Figure 6. Half of the sample was treated with RNaseH in vitro before the …
Figure 7
Proposed model of R-loop processing.
Polymerase stalling at sites of nicks, topoisomerase adducts (TopI adduct shown here on non-template strand), or other lesions generates a nascent R-loop structure (RNA shown in red). Cleavage of …
Figure 8
CtIP and its nuclease activity promote ssDNA break formation.
(A) DNA breaks were quantified in wild-type and CtIP-depleted U2OS cells by alkaline comet assay. Cells were untreated (DMSO) or exposed to 5 µM CPT for 1 hr, with DRB (20 µM) or aphidicolin (2 …
Author response image 1
Overexpression of RNH1 or SEN1 does not suppress the sensitivity of Δsae2 strains to hydroxyurea (HU) exposure.
(A). RNH1 was induced from the GAL1 promoter on a CEN plasmid in Δsae2 compared to Δsae2 with vector only. Viability with or without HU (100 mM) was assessed at 48 hours or 72 hours as indicated. …
Author response image 2
CtIP, XPG, or Senataxin-depleted cells exhibit higher levels of RNA-DNA hybrids.
(A) Human U2OS cells expressing shRNA specific for CtIP with or without RNaseH expression as indicated were analyzed using immunofluorescence with S9.6 antibody and with antinucleolin antibody which …
Author response image 3
Browser view of Sae2-ChIP at the GPR1, TBS1, and SAM2 genes in sae2Δ cells expressing Flag-Sae2, in S phase with CPT exposure compared to S phase with no DNA damage.
Reads from the immunoprecipitated sample are shown (IP) in comparison to control immunoprecipitations performed in the absence of Flag antibody (bead control, BC).
Author response image 4
Browser views of Sae2-ChIP at the rDNA locus and the PDC1 locus on ch.XII in sae2Δ cells expressing Flag-Sae2, in S phase with CPT exposure compared to S phase with no DNA damage.
https://doi.org/10.7554/eLife.42733.027Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (yeast) | yeast strains see supplementary file 1 | |||
| Cell line (human) | U2OS | ATCC | ||
| Cell line (human) | HEK-293T | ATCC | ||
| Cell line (human) | U2OS T-RexTM Flp-in | Jeff Parvin | U2OS Flp-in | Invitrogen tet-on system |
| Transfected construct (human) | pTP3146 | this study | GFP-CtIP | see Materials and methods |
| Transfected construct (human) | pTP3663 | this study | Flag-Bio-CtIP | see Materials and methods |
| Transfected construct (human) | pTP3665 | this study | Flag-Bio-CtIP(N289A/H290A) | see Materials and methods |
| Transfected construct (human) | pTP3531 | this study | C-term of Senataxin in pcDNA5/FRT/TO | see Materials and methods |
| Transfected construct (human) | pTP4195 | this study | PCF11 in pcDNA5/FRT/TO | see Materials and methods |
| Transfected construct (E. Coli) | pTP3492 | this study | wild-type RNaseH-mCherry in pcDNA5/FRT/TO | see Materials and methods |
| Transfected construct | pTP3494 | this study | mCherry only in pcDNA5/FRT/TO | see Materials and methods |
| Transfected construct (E. Coli) | pTP3493 | this study | RNaseHI-D10R-E48R in pcDNA5/FRT/TO | see Materials and methods |
| Transfected construct (E. Coli) | pTP3660 | this study | RNaseHI-D10R- E48R in lentivirus vector | see Materials and methods |
| Transfected construct | pRSITEP--U6Tet-(sh)-EF1-TetRep-2A-shRNA-CtIP | Cellecta | inducible shRNA for CtIP | see Materials and methods |
| Transfected construct | pTP3703 | this study | inducible shRNA for XPG | see Materials and methods |
| Transfected construct | pTP3677 | this study | inducible shRNA for SETX | see Materials and methods |
| Transfected construct | pTP4195 | this study | Sγ3 sequence in pcDNA5/FRT/TO | see Materials and methods |
| Other | primers see supplementary file 4 |
Additional files
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Supplementary file 1
Yeast strain list.
- https://doi.org/10.7554/eLife.42733.018
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Supplementary file 2
Sae2 ChIP-seq data.
- https://doi.org/10.7554/eLife.42733.019
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Supplementary file 3
CtIP RNA-seq data.
- https://doi.org/10.7554/eLife.42733.020
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Supplementary file 4
Oligonucleotides.
- https://doi.org/10.7554/eLife.42733.021
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Transparent reporting form
- https://doi.org/10.7554/eLife.42733.022
