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Involvement of ATM in homologous recombination after end resection and RAD51 nucleofilament formation.

Bakr A, Oing C, Köcher S, Borgmann K, Dornreiter I, Petersen C, Dikomey E, Mansour WY - Nucleic Acids Res. (2015)

Bottom Line: Here we show for the first time that ATM is also needed for later steps in HR after RAD51 nucleofilament formation.Moreover, we demonstrated that ATR can partly compensate for the deficiency in early, but not in later, steps of HR upon ATM inhibition.Taken together, we describe here for the first time that ATM is needed not only for the initiation but also for the completion of HR.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Radiobiology & Experimental Radiooncology, University Medical Center Hamburg-Eppendorf, Hamburg 20246, Germany.

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ATM inhibition after end resection leads to HR deficiency and increases radiosensitization after PARP inhibition. (A) Upper panel: Schematic representation of the inducible I-SceI-expressing vector pDSRed-I-SceI-GR. Addition of triamcinolone (TA) leads to the nuclear localization of the I-SceI enzyme. Lower panel: HeLa cells harboring the pGC reporter were transfected with pDSRed-I-SceI-GR. Twenty-four hours later, MRE11 or ATM was inhibited either 2 h prior to TA treatment (pre) or 2 , 6 or 12 h after TA (post). GFP+ cells were evaluated 48 h after I-SceI-transfection as an indication of HR efficiency. (B) Radiosensitivity of exponentially growing A549 cells was measured by colony forming assay after inhibition of ATM (10-μM KU55933) and/or PARP (1-μM Olaparib) either prior to (-pre) or after (-post) the indicated X-ray doses. Error bars represent the SEM of three independent experiments.
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Figure 5: ATM inhibition after end resection leads to HR deficiency and increases radiosensitization after PARP inhibition. (A) Upper panel: Schematic representation of the inducible I-SceI-expressing vector pDSRed-I-SceI-GR. Addition of triamcinolone (TA) leads to the nuclear localization of the I-SceI enzyme. Lower panel: HeLa cells harboring the pGC reporter were transfected with pDSRed-I-SceI-GR. Twenty-four hours later, MRE11 or ATM was inhibited either 2 h prior to TA treatment (pre) or 2 , 6 or 12 h after TA (post). GFP+ cells were evaluated 48 h after I-SceI-transfection as an indication of HR efficiency. (B) Radiosensitivity of exponentially growing A549 cells was measured by colony forming assay after inhibition of ATM (10-μM KU55933) and/or PARP (1-μM Olaparib) either prior to (-pre) or after (-post) the indicated X-ray doses. Error bars represent the SEM of three independent experiments.

Mentions: To further confirm that the inhibition of ATM following end resection results in HR deficiency, we made use of the pDSRed-I-SceI-GR plasmid (29) (Figure 5A, upper panel), which expresses an I-SceI endonuclease fused to a DSRed domain. I-SceI is also fused to a glucocorticoid receptor (GR) ligand-binding domain, which allows for the nuclear localization of the plasmid upon addition of triamcinolone acetonide (TA). Having established that MRE11 is involved exclusively in the end resection step, we first sought to determine the exact length of time that MRE11 is actually needed for HR, a time point which indicates the completion of end resection. To this end, HeLa cells carrying pGC were transfected with pDSRed-ISceI-GR and 24 h later, MRE11 nuclease activity was inhibited either 1 h prior to or 2, 6 and 12 h after TA treatment. As shown in Figure 5A, HR was significantly suppressed upon inhibition of MRE11 prior to TA treatment (0.05 ± 0.009 compared to 0.218 ± 0.01; P = 0.001). The inhibitory effect on HR was partly relieved when MRE11 was inhibited 2 and 6 h post-TA treatment, and was completely abrogated when MRE11 was inhibited 12 h after exposure to TA. These data indicate that DSB end resection by MRE11 is fully completed 12 h after the addition of TA. Importantly, when ATM was inhibited at the 12-h time point following treatment with TA, HR was still found to be reduced by more than 50% (Figure 5A, black columns). In line with the data in Figure 1, ATM inhibition prior to DSB induction strongly reduced HR. Overall, these data clearly confirm that in contrast to MRE11, ATM continues to be involved in HR after DSB end resection.


Involvement of ATM in homologous recombination after end resection and RAD51 nucleofilament formation.

Bakr A, Oing C, Köcher S, Borgmann K, Dornreiter I, Petersen C, Dikomey E, Mansour WY - Nucleic Acids Res. (2015)

ATM inhibition after end resection leads to HR deficiency and increases radiosensitization after PARP inhibition. (A) Upper panel: Schematic representation of the inducible I-SceI-expressing vector pDSRed-I-SceI-GR. Addition of triamcinolone (TA) leads to the nuclear localization of the I-SceI enzyme. Lower panel: HeLa cells harboring the pGC reporter were transfected with pDSRed-I-SceI-GR. Twenty-four hours later, MRE11 or ATM was inhibited either 2 h prior to TA treatment (pre) or 2 , 6 or 12 h after TA (post). GFP+ cells were evaluated 48 h after I-SceI-transfection as an indication of HR efficiency. (B) Radiosensitivity of exponentially growing A549 cells was measured by colony forming assay after inhibition of ATM (10-μM KU55933) and/or PARP (1-μM Olaparib) either prior to (-pre) or after (-post) the indicated X-ray doses. Error bars represent the SEM of three independent experiments.
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Figure 5: ATM inhibition after end resection leads to HR deficiency and increases radiosensitization after PARP inhibition. (A) Upper panel: Schematic representation of the inducible I-SceI-expressing vector pDSRed-I-SceI-GR. Addition of triamcinolone (TA) leads to the nuclear localization of the I-SceI enzyme. Lower panel: HeLa cells harboring the pGC reporter were transfected with pDSRed-I-SceI-GR. Twenty-four hours later, MRE11 or ATM was inhibited either 2 h prior to TA treatment (pre) or 2 , 6 or 12 h after TA (post). GFP+ cells were evaluated 48 h after I-SceI-transfection as an indication of HR efficiency. (B) Radiosensitivity of exponentially growing A549 cells was measured by colony forming assay after inhibition of ATM (10-μM KU55933) and/or PARP (1-μM Olaparib) either prior to (-pre) or after (-post) the indicated X-ray doses. Error bars represent the SEM of three independent experiments.
Mentions: To further confirm that the inhibition of ATM following end resection results in HR deficiency, we made use of the pDSRed-I-SceI-GR plasmid (29) (Figure 5A, upper panel), which expresses an I-SceI endonuclease fused to a DSRed domain. I-SceI is also fused to a glucocorticoid receptor (GR) ligand-binding domain, which allows for the nuclear localization of the plasmid upon addition of triamcinolone acetonide (TA). Having established that MRE11 is involved exclusively in the end resection step, we first sought to determine the exact length of time that MRE11 is actually needed for HR, a time point which indicates the completion of end resection. To this end, HeLa cells carrying pGC were transfected with pDSRed-ISceI-GR and 24 h later, MRE11 nuclease activity was inhibited either 1 h prior to or 2, 6 and 12 h after TA treatment. As shown in Figure 5A, HR was significantly suppressed upon inhibition of MRE11 prior to TA treatment (0.05 ± 0.009 compared to 0.218 ± 0.01; P = 0.001). The inhibitory effect on HR was partly relieved when MRE11 was inhibited 2 and 6 h post-TA treatment, and was completely abrogated when MRE11 was inhibited 12 h after exposure to TA. These data indicate that DSB end resection by MRE11 is fully completed 12 h after the addition of TA. Importantly, when ATM was inhibited at the 12-h time point following treatment with TA, HR was still found to be reduced by more than 50% (Figure 5A, black columns). In line with the data in Figure 1, ATM inhibition prior to DSB induction strongly reduced HR. Overall, these data clearly confirm that in contrast to MRE11, ATM continues to be involved in HR after DSB end resection.

Bottom Line: Here we show for the first time that ATM is also needed for later steps in HR after RAD51 nucleofilament formation.Moreover, we demonstrated that ATR can partly compensate for the deficiency in early, but not in later, steps of HR upon ATM inhibition.Taken together, we describe here for the first time that ATM is needed not only for the initiation but also for the completion of HR.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Radiobiology & Experimental Radiooncology, University Medical Center Hamburg-Eppendorf, Hamburg 20246, Germany.

Show MeSH
Related in: MedlinePlus