A local ATR-dependent checkpoint pathway is activated by a site-specific replication fork block in human cells
- Department of Radiation Oncology and the Molecular Biology Program, Memorial Sloan Kettering Cancer Center, United States
- Department of Cell Biology, Albert Einstein College of Medicine, United States
- Molecular Biology Program, Memorial Sloan Kettering Cancer Center, United States
Figures
Figure 1 with 1 supplement
Tus is bound to TerB sites integrated in human cells.
(A) Schematic of linearized plasmid (pWB15) integrated into MCF7 cells (MCF7 5C-TerB) with two cassettes containing five tandem TerB sequences in the non-permissive orientation (black arrows). Black …
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Figure 1—source data 1
Tables related to Figure 1B and E.
- https://cdn.elifesciences.org/articles/87357/elife-87357-fig1-data1-v1.zip
Figure 1—figure supplement 1
Generation of the MCF7 5C-TerB cell line.
Schematic of the integration of pWB15 in MCF7 cells to generate MCF7 5C-TerB. pWB15 contains two TerB cassettes (gray arrows). Each TerB cassette contains five tandem TerB sequences, which are in …
Figure 2 with 2 supplements
Replication fork pauses at the Ter sequence in the presence of the Tus protein.
(A) Schematic of pulse labeling of MCF7 5C-TerB cells with Tus protein expression for 3 d. (B) Locus map of a 200 kb SfiI segment from MCF7 5C-TerB Chromosome 12 with the integrated plasmid (pWB15) …
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Figure 2—source data 1
Table related to Figure 2H.
- https://cdn.elifesciences.org/articles/87357/elife-87357-fig2-data1-v1.zip
Figure 2—figure supplement 1
Expression of Tus in MCF7 5C-TerB cells.
(A) Schematic of doxycycline (Dox)-inducible expression of Myc-NLS-TUS-SNAP. (rtTA = reverse tetracycline trans activator; TRE = tetracycline response element). (B) Immunoblot of MCF7 5C-TerB cells …
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Figure 2—figure supplement 1—source data 1
Unedited western blot images for Figure 2—figure supplement 1B.
- https://cdn.elifesciences.org/articles/87357/elife-87357-fig2-figsupp1-data1-v1.zip
Figure 2—figure supplement 2
Enrichment of FANCM at the Ter sequence in the presence of the Tus protein.
Chromatin immunoprecipitation (ChIP) using FANCM antibody was performed MCF7 5C-TerB cells transfected with vector control (VC) or HA-Tus-His plasmids. ChIP-qPCR was conducted using the indicated …
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Figure 2—figure supplement 2—source data 1
Table related Figure 2—figure supplement 2.
- https://cdn.elifesciences.org/articles/87357/elife-87357-fig2-figsupp2-data1-v1.zip
Figure 3 with 2 supplements
γH2AX is enriched at TerB sites after Tus expression.
(A) MCF7 5C-TerB cells transfected with GFP or GFP-Tus, γH2AX and IgG antibodies were used to immuno-precipitate proteins and analyzed by immunoblotting with indicated antibodies. (B) Schematic of …
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Figure 3—source data 1
Unedited western blot images for Figure 3A.
- https://cdn.elifesciences.org/articles/87357/elife-87357-fig3-data1-v1.zip
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Figure 3—source data 2
Tables related to Figure 3D and F.
- https://cdn.elifesciences.org/articles/87357/elife-87357-fig3-data2-v1.zip
Figure 3—figure supplement 1
Distinct patterns of γH2AX enrichment at a Cas9-mediated DSB versus the Tus-TerB fork barrier.
(A) Schematic of the linearized TerB plasmid (pWB15) integrated as a unique copy into MCF7 cells (MCF7 5C-TerB) with the Cas9-binding site (green protein). Blue triangles: Cas9 cut site. Black …
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Figure 3—figure supplement 1—source data 1
Unedited agarose gel for Figure 1—figure supplement 1C.
- https://cdn.elifesciences.org/articles/87357/elife-87357-fig3-figsupp1-data1-v1.zip
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Figure 3—figure supplement 1—source data 2
Table related to Figure 3—figure supplement 1D.
- https://cdn.elifesciences.org/articles/87357/elife-87357-fig3-figsupp1-data2-v1.zip
Figure 3—figure supplement 2
Genome-wide gH2AX foci were unaffected with Tus expression.
(A) Representative images of gH2AX foci across stated conditions. Scale Bars, 10 μm. (B) Average number of gH2AX foci per nucleus in conditions stated. Cells treated with or without 2 mM …
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Figure 3—figure supplement 2—source data 1
Table related to Figure 3—figure supplement 2B.
- https://cdn.elifesciences.org/articles/87357/elife-87357-fig3-figsupp2-data1-v1.zip
Figure 4 with 1 supplement
γH2AX phosphorylation at Tus-TerB is ATR-dependent.
(A) MCF7 5C-TerB cells transfected with vector control (VC) or HA-Tus were treated for 4 hr with 2 mM hydroxyurea (HU) before lysis and fractionation (whole-cell extract [WCE], soluble fraction, and …
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Figure 4—source data 1
Unedited western blot images for Figure 4A.
- https://cdn.elifesciences.org/articles/87357/elife-87357-fig4-data1-v1.zip
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Figure 4—source data 2
Unedited western blot images for Figure 4B.
- https://cdn.elifesciences.org/articles/87357/elife-87357-fig4-data2-v1.zip
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Figure 4—source data 3
Tables related to Figure 4D and E.
- https://cdn.elifesciences.org/articles/87357/elife-87357-fig4-data3-v1.zip
Figure 4—figure supplement 1
Cell cycle progression was unaffected by Tus expression.
(A) Cell cycle analysis and (B) quantification was performed using flow cytometry following staining with propidium iodide staining in MCF7 5C-TerB cells under the stated conditions. Cells treated …
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Figure 4—figure supplement 1—source data 1
Table related to Figure 4—figure supplement 1B.
- https://cdn.elifesciences.org/articles/87357/elife-87357-fig4-figsupp1-data1-v1.zip
Figure 5
A local ATR-dependent checkpoint is activated by the Tus-TerB replication fork barrier (RFB).
A model depicting the cellular response to a site-specific DSB using CRISPR-Cas9 and a global replication stress with hydroxyurea, both of which lead to a DNA damage response (DDR, yellow …
Additional files
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Supplementary file 1
Supplementary information for Figure 2 and extended methods.
(a) Demonstration that the Tus-Ter replication fork block does not activate significant replication elsewhere in the genome. Replication characteristics of 200 kb global DNA segments that represent the total genome. These measurements do not include the segments containing the TerB sequence. The table compares MCF7 cells containing the TerB sequence with and without Tus induced. This was determined on stretched DNA molecules that had completely incorporated IdU, CldU, or a combination of both nucleotide analogs. (b) List of antibodies, oligos, and plasmids used in the study.
- https://cdn.elifesciences.org/articles/87357/elife-87357-supp1-v1.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/87357/elife-87357-mdarchecklist1-v1.pdf
