Synergistic Roles of Non-Homologous End Joining and Homologous Recombination in Repair of Ionizing Radiation-Induced DNA Double Strand Breaks in Mouse Embryonic Stem Cells
<p>mES cells lacking DNA-PK<sub>cs</sub> and Rad54 are hypersensitive to X-ray radiation. (<b>a</b>) Two independent clones of the DNA-PK<sub>cs</sub><sup>−/−</sup> Rad54<sup>−/−</sup> mES cell line were generated in a two-step process. First, DNA-PK<sub>cs</sub><sup>−/−</sup> mES cells were targeted with a targeting construct against Rad54 containing a hygromycin resistance gene to generate DNA-PK<sub>cs</sub><sup>−/−</sup> Rad54<sup>WT/−</sup> mES cells. Secondly, two independent DNA-PK<sub>cs</sub><sup>−/−</sup> Rad54<sup>WT/−</sup> mES cell clones were targeted with a targeting construct against Rad54 containing a puromycin resistance gene to generate DNA-PK<sub>cs</sub><sup>−/−</sup> Rad54<sup>−/−</sup> mES cells. (<b>b</b>) Western blot was used to confirm the lack of Rad54 and DNA-PK<sub>cs</sub> in the mES cells with the indicated genotypes. The upper Western blot in the figure shows probing for Rad54 and β-actin as the loading control. The lower Western blot in the figure shows probing for DNA-PK<sub>cs</sub> and vinculin as the loading control. (<b>c</b>) Clonogenic survival of mES cell lines with indicated genotypes after X-ray irradiation. Error bars represent SEM.</p> "> Figure 2
<p>mES cells lacking DNA-PK<sub>cs</sub> and Rad54 show impaired resolution of 53BP1 foci and increased nuclear size after 2 Gy of X-ray radiation. (<b>a</b>) Representative images of mES cells irradiated with 2 Gy of X-ray radiation and incubated for indicated times. After the recovery time, cells were fixed and stained for 53BP1. (<b>b</b>) Quantification of 53BP1 foci per mES nucleus (left) and area of mES nuclei (right). Error bars represent SEM.</p> "> Figure 3
<p>X-ray irradiation results in more persistent G2 phase cell cycle block in mES cells lacking DNA-PK<sub>cs</sub> and Rad54. (<b>a</b>) mES cells were irradiated with 1 Gy of X-ray radiation and incubated for indicated times. After recovery time, cells were fixed and stained for DAPI (DNA content), EdU (S phase cells), and phospho-H3 (mitotic (M) phase cells). Cell cycle distribution was analyzed using flow cytometry. (<b>b</b>) Quantification of percentage of G1, S, G2, and M phase cells in mES cells irradiated with 1 Gy of X-ray radiation, as shown in (<b>a</b>). (<b>c</b>) mES cells were irradiated with 1 and 2 Gy of X-ray radiation and incubated for indicated times. After recovery time, cells were fixed, and DNA was stained using Propidium Iodide. Cell cycle distribution was analyzed using flow cytometry. (<b>d</b>) Quantification of percentage of G1, S, and G2 phase cells in mES cells irradiated with 1 and 2 Gy of X-ray radiation, as shown in (<b>c</b>).</p> "> Figure 3 Cont.
<p>X-ray irradiation results in more persistent G2 phase cell cycle block in mES cells lacking DNA-PK<sub>cs</sub> and Rad54. (<b>a</b>) mES cells were irradiated with 1 Gy of X-ray radiation and incubated for indicated times. After recovery time, cells were fixed and stained for DAPI (DNA content), EdU (S phase cells), and phospho-H3 (mitotic (M) phase cells). Cell cycle distribution was analyzed using flow cytometry. (<b>b</b>) Quantification of percentage of G1, S, G2, and M phase cells in mES cells irradiated with 1 Gy of X-ray radiation, as shown in (<b>a</b>). (<b>c</b>) mES cells were irradiated with 1 and 2 Gy of X-ray radiation and incubated for indicated times. After recovery time, cells were fixed, and DNA was stained using Propidium Iodide. Cell cycle distribution was analyzed using flow cytometry. (<b>d</b>) Quantification of percentage of G1, S, and G2 phase cells in mES cells irradiated with 1 and 2 Gy of X-ray radiation, as shown in (<b>c</b>).</p> "> Figure 4
<p>mES cells lacking DNA-PK<sub>cs</sub> and expressing ATPase-defective Rad54 are hypersensitive to X-ray radiation. (<b>a</b>) The DNA-PK<sub>cs</sub><sup>−/−</sup> Rad54<sup>WT-GFP/−</sup> and DNA-PK<sub>cs</sub><sup>−/−</sup> Rad54<sup>KR-GFP/−</sup> mES cell lines were generated by targeting the mES DNA-PK<sub>cs</sub><sup>−/−</sup> Rad54<sup>WT/−</sup> 1 cell line with a targeting construct against Rad54 containing Rad54<sup>WT-GFP</sup> or Rad54<sup>KR-GFP</sup>. (<b>b</b>) Western blot was used to confirm the knockin of GFP-Rad54 in mES cells with the indicated genotypes. The upper Western blot in the figure shows probing for Rad54 and β-actin as the loading control. The lower Western blot in the figure shows probing for DNA-PKcs and vinculin as the loading control. (<b>c</b>) Sanger sequencing results to confirm K189R mutation in Rad54. (<b>d</b>) Clonogenic survival of mES cell lines with indicated genotypes after X-ray irradiation. Error bars represent SEM.</p> "> Figure 5
<p>mES cells lacking DNA-PK<sub>cs</sub> show impaired Rad54 focus resolution and increased nuclear size after 2 Gy of X-ray radiation. (<b>a</b>) Representative images of mES cells irradiated with 2 Gy of X-ray radiation and incubated for indicated times. After the recovery time, cells were fixed and imaged for Rad54. (<b>b</b>) Quantification of Rad54 foci per mES nucleus. Rad54 foci disappear in cells with enlarged nuclei (<a href="#cells-13-01462-f006" class="html-fig">Figure 6</a>); therefore, nuclei larger than 400 µm<sup>2</sup> were excluded from the analysis. Error bars represent SEM. (<b>c</b>) Quantification of area of mES nuclei. Error bars represent SEM.</p> "> Figure 6
<p>Rad54 foci disappear in mitotic cells and in cells with enlarged nuclei. (<b>a</b>) Representative images of live-cell imaging of WT Rad54<sup>WT-GFP/−</sup> (top) and DNA-PK<sub>cs</sub><sup>−/−</sup> Rad54<sup>WT-GFP/−</sup> (bottom) mES cells. Top row shows the cells going through cell division with the disappearance of Rad54 foci towards cell division. Bottom row shows the cell with disappearing Rad54 foci, swelling up but with no cell division happening. (<b>b</b>) Quantification of the Rad54-GFP foci in the cells shown in (<b>a</b>). (<b>c</b>) Quantification of the percentage of dividing cells in the live-cell image. (<b>d</b>) Quantification of the percentage of cells that show a larger nuclear size.</p> "> Figure 7
<p>Increased micronuclei formation in mES cells lacking DNA-PK<sub>cs</sub> and expressing ATPase-defective Rad54. (<b>a</b>) Representative DAPI images of WT Rad54<sup>WT-GFP/−</sup>, WT Rad54<sup>KR-GFP/−</sup>, DNA-PK<sub>cs</sub><sup>−/−</sup> Rad54<sup>WT-GFP/−</sup>, and DNA-PK<sub>cs</sub><sup>−/−</sup> Rad54<sup>KR-GFP/−</sup> mES cells 24 h after 2 Gy of X-ray radiation. (<b>b</b>) Quantification of the percentage of cells that have micronuclei in fixed samples. Error bars represent SEM. Asterisks represent the following <span class="html-italic">p</span>-values: * ≤ 0.05; ** ≤ 0.01 (left). Quantification of the percentage of cells that are normal, have micronuclei, or have chromatin bridges in live-cell imaging data (right). (<b>c</b>) Number of micronuclei per cell in fixed samples (left, normalized for total number of nuclei) and live-cell imaging data (right, normalized for total number of cell divisions).</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. mES Cell Culture
2.2. Generation of mES DNA-PKcs−/− Rad54−/− and DNA-PKcs−/− Rad54GFP-Knockin Cell Lines
2.3. Western Blotting
2.4. Irradiation
2.5. Clonogenic Survival
2.6. Immunofluorescence Staining
2.7. Microscopy
2.8. Live-Cell Imaging of Rad54-GFP
2.9. Cell Cycle Analysis
2.10. Statistics
3. Results
3.1. mES Cells Lacking DNA-Pkcs and Rad54 Are Hypersensitive to X-ray Radiation
3.2. mES Lacking DNA-PKcs and Rad54 Cells Show Impaired 53BP1 Focus Resolution and an Increased Nuclear Size after 2 Gy of X-ray Radiation
3.3. X-ray Irradiation Results in More Persistent G2 Phase Cell Cycle Block in mES Cells Lacking DNA-PKcs and Rad54
3.4. mES Cells Lacking DNA-PKcs and Rad54 or Expressing ATPase-Defective Rad54 Show Similar Sensitivity to X-Ray Radiation
3.5. mES Cells Lacking DNA-PKcs Show Elevated Levels of Rad54 Foci after X-ray Irradiation
3.6. Increased Genomic Instability in Cells Lacking DNA-PKcs and Expressing ATPase-Defective Rad54
4. Discussion
4.1. Increased Nuclear Size of mES Cells Lacking DNA-PKcs
4.2. The Role of DNA-PKcs and Rad54 in Cell Cycle Regulation and Genome Stability Maintenance in mES Cells
4.3. Enhanced Activity of Rad54 in the Absence of DNA-PKcs
4.4. Therapeutic Applications
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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van de Kamp, G.; Heemskerk, T.; Kanaar, R.; Essers, J. Synergistic Roles of Non-Homologous End Joining and Homologous Recombination in Repair of Ionizing Radiation-Induced DNA Double Strand Breaks in Mouse Embryonic Stem Cells. Cells 2024, 13, 1462. https://doi.org/10.3390/cells13171462
van de Kamp G, Heemskerk T, Kanaar R, Essers J. Synergistic Roles of Non-Homologous End Joining and Homologous Recombination in Repair of Ionizing Radiation-Induced DNA Double Strand Breaks in Mouse Embryonic Stem Cells. Cells. 2024; 13(17):1462. https://doi.org/10.3390/cells13171462
Chicago/Turabian Stylevan de Kamp, Gerarda, Tim Heemskerk, Roland Kanaar, and Jeroen Essers. 2024. "Synergistic Roles of Non-Homologous End Joining and Homologous Recombination in Repair of Ionizing Radiation-Induced DNA Double Strand Breaks in Mouse Embryonic Stem Cells" Cells 13, no. 17: 1462. https://doi.org/10.3390/cells13171462