Mofidi, Amir (2017)
DNA-double strand break repair and cell cycle control of murine stem cells after exposure to ionizing radiation.
Technische Universität Darmstadt
Dissertation, Erstveröffentlichung
Kurzbeschreibung (Abstract)
Ionizing radiation (IR) induces a variety of DNA lesions among which DNA double strand breaks (DSBs) are biologically most significant. In somatic cells, several cellular DNA damage response (DDR) mechanisms such as cell cycle checkpoints and DSB repair pathways work in concern to handle these threats. In response to DNA damage, G1/S and G2/M checkpoints activities prevent the progression of the cells to the next cell cycle phase. This mechanism prohibits replication and division of the cells containing DSBs and provides them time for repair. In parallel to this event, DNA repair machinery repairs the DSBs. The majority of IR-induced DSBs are repaired fast via canonical non-homologous end-joining (c-NHEJ) in which DNA-PKcs is one of the core enzymes. In contrast, a sub-fraction of breaks is repaired with slow kinetics in an ATM-dependent manner. This repair pathway represents homologous recombination (HR) in G2 and resection-dependent c-NHEJ in G1 phase. In stem cells, although it is appreciated that DDR regulation is distinct from that in somatic cells, the key factors and their functional mechanisms still remain unknown. The main aim of this thesis was to understand the mechanism/s by which stem cells retain their genomic integrity. Moreover, the level of repair capacity in pluripotent and multipotent stem cells has been compared. To achieve these aims, the DDR mechanism has been characterized in mouse embryonic stem cells (ESCs) and ESC-derived neural stem cell (NSCs). Cell cycle checkpoint analysis after 2 Gy X-rays demonstrated an ineffective G1/S checkpoint arrest in NSCs, whereas, ESCs failed to prevent cell cycle progression into S phase. In both cell types, cell cycle was completely arrested by G2/M checkpoint. However, ESCs showed a prolonged G2/M arrest compared to NSCs. Analyzing DSB repair in NSCs after exposure to 10 mGy and 100 mGy X-rays revealed that the repair capacity is reduced by decreasing the radiation dose. After 10 mGy IR, the value of the IR-induced DSBs was remained constant until 4 h post IR. This is evident that the DSB repair machinery cannot be fully activated by low doses of IR. Investigation of DSB repair capacity after 2 Gy X-rays showed that wild type (WT) ESCs and NSCs have similar repair kinetics and almost all IR-induced DSBs were repaired within 6 h post IR. Inhibition of ATM impaired the slow component of DSB repair in both cell types, whereas, the fast component was not affected. Interestingly, DNA-PKcs inhibitor induced a temporary repair defect followed by an efficient repair to the background DSB levels in ESCs. In contrast, in NSCs, DSB repair was almost stalled after inhibition of DNA-PKcs. Moreover, inhibition of Rad51 impaired the DSB repair in G2-phase NSCs, whereas in ESCs, the repair kinetic was not impaired. Therefore, we asked if an alternative repair pathway provides a backup repair mechanism in DNA-PKcs- and Rad51-deficient ESCs. To address the aforementioned question, we investigated the role of alt-NHEJ pathway in ESCs and NSCs by inhibiting PARP1. Importantly, PARP1-inhibition did not influence DSB repair kinetics in WT or ATM-inhibited cells. However, inhibition of PARP1 induced an additional significant repair defect in DNA-PKcs- and Rad51-inhibited ESCs, not in NSCs. These data demonstrated that PARP1-dependent alt-NHEJ functions as a backup repair pathway for impaired c-NHEJ or HR in ESCs. It is well know that PARP1-dependent alt-NHEJ is a resection dependent pathway. Analyzing resection in G2 phase by scoring Rad51 foci displayed a higher foci level in ESCs than in NSCs. In addition, investigation of resection in G1 phase uncovered that ESCs form pRPA foci, not NSCs. Furthermore, inhibition of the proteins regulating resection in G1 phase, like PLK3, not only diminished the formation of pRPA foci but also impaired DSB repair in ESCs. Whereas, in non-pluripotent cells (NSCs or HeLa cells), no repair defect was observed after inhibition of resection in G1 phase. These observations revealed that ESCs perform more resection than NSCs, suggesting that resection dependent-NHEJ is a prominent DSB repair pathways in G1-phase ESCs. All together, the data implies that, ESCs can perform long-range resection of DSB ends and, therefore, in case of impaired classical repair pathways, can readily switch to PARP1-dependent alt-NHEJ. Previously, it was shown that nascent RNAs mediate an error-free c-NHEJ by serving as templates to faithfully restore the lost genomic information at the break site. Moreover, it was demonstrated that RNA transcription machinery functions as a molecular motor to promote excessive DNA resection. These evidences led to the curiosity to understand if RNA mediates the long range resection in G1-phase ESCs. The detection of RNA-DNA hybrids at the DSB sites, as well as, reduction in pRPA foci level after inhibition of transcription verified our hypothesis that RNA transcription machinery might mediate long-range resection in G1 phase ESCs. Furthermore, destabilization of the RNA-DNA hybrids by overexpression of RNaseH1 enzyme, induced a significant repair defect in G1 phase ESCs. This effect was identical to the repair defect which was observed after inhibition of PLK3. These observations confirmed the role of RNA in mediating resection in G1-phase ESCs. These findings suggest that, the involvement of RNA as a template during DSB repair might be mediating an error-free repair and thus play an important role in maintaining the genomic integrity of pluripotent stem cells.
Typ des Eintrags: | Dissertation | ||||
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Erschienen: | 2017 | ||||
Autor(en): | Mofidi, Amir | ||||
Art des Eintrags: | Erstveröffentlichung | ||||
Titel: | DNA-double strand break repair and cell cycle control of murine stem cells after exposure to ionizing radiation | ||||
Sprache: | Englisch | ||||
Referenten: | Löbrich, Prof. Dr. Markus ; Nuber, Prof. Dr. Ulrike ; Laube, Prof. Dr. Bodo ; Rödel, Prof. Dr. Franz | ||||
Publikationsjahr: | 14 November 2017 | ||||
Ort: | Darmstadt | ||||
Datum der mündlichen Prüfung: | 14 November 2017 | ||||
URL / URN: | http://tuprints.ulb.tu-darmstadt.de/6975 | ||||
Kurzbeschreibung (Abstract): | Ionizing radiation (IR) induces a variety of DNA lesions among which DNA double strand breaks (DSBs) are biologically most significant. In somatic cells, several cellular DNA damage response (DDR) mechanisms such as cell cycle checkpoints and DSB repair pathways work in concern to handle these threats. In response to DNA damage, G1/S and G2/M checkpoints activities prevent the progression of the cells to the next cell cycle phase. This mechanism prohibits replication and division of the cells containing DSBs and provides them time for repair. In parallel to this event, DNA repair machinery repairs the DSBs. The majority of IR-induced DSBs are repaired fast via canonical non-homologous end-joining (c-NHEJ) in which DNA-PKcs is one of the core enzymes. In contrast, a sub-fraction of breaks is repaired with slow kinetics in an ATM-dependent manner. This repair pathway represents homologous recombination (HR) in G2 and resection-dependent c-NHEJ in G1 phase. In stem cells, although it is appreciated that DDR regulation is distinct from that in somatic cells, the key factors and their functional mechanisms still remain unknown. The main aim of this thesis was to understand the mechanism/s by which stem cells retain their genomic integrity. Moreover, the level of repair capacity in pluripotent and multipotent stem cells has been compared. To achieve these aims, the DDR mechanism has been characterized in mouse embryonic stem cells (ESCs) and ESC-derived neural stem cell (NSCs). Cell cycle checkpoint analysis after 2 Gy X-rays demonstrated an ineffective G1/S checkpoint arrest in NSCs, whereas, ESCs failed to prevent cell cycle progression into S phase. In both cell types, cell cycle was completely arrested by G2/M checkpoint. However, ESCs showed a prolonged G2/M arrest compared to NSCs. Analyzing DSB repair in NSCs after exposure to 10 mGy and 100 mGy X-rays revealed that the repair capacity is reduced by decreasing the radiation dose. After 10 mGy IR, the value of the IR-induced DSBs was remained constant until 4 h post IR. This is evident that the DSB repair machinery cannot be fully activated by low doses of IR. Investigation of DSB repair capacity after 2 Gy X-rays showed that wild type (WT) ESCs and NSCs have similar repair kinetics and almost all IR-induced DSBs were repaired within 6 h post IR. Inhibition of ATM impaired the slow component of DSB repair in both cell types, whereas, the fast component was not affected. Interestingly, DNA-PKcs inhibitor induced a temporary repair defect followed by an efficient repair to the background DSB levels in ESCs. In contrast, in NSCs, DSB repair was almost stalled after inhibition of DNA-PKcs. Moreover, inhibition of Rad51 impaired the DSB repair in G2-phase NSCs, whereas in ESCs, the repair kinetic was not impaired. Therefore, we asked if an alternative repair pathway provides a backup repair mechanism in DNA-PKcs- and Rad51-deficient ESCs. To address the aforementioned question, we investigated the role of alt-NHEJ pathway in ESCs and NSCs by inhibiting PARP1. Importantly, PARP1-inhibition did not influence DSB repair kinetics in WT or ATM-inhibited cells. However, inhibition of PARP1 induced an additional significant repair defect in DNA-PKcs- and Rad51-inhibited ESCs, not in NSCs. These data demonstrated that PARP1-dependent alt-NHEJ functions as a backup repair pathway for impaired c-NHEJ or HR in ESCs. It is well know that PARP1-dependent alt-NHEJ is a resection dependent pathway. Analyzing resection in G2 phase by scoring Rad51 foci displayed a higher foci level in ESCs than in NSCs. In addition, investigation of resection in G1 phase uncovered that ESCs form pRPA foci, not NSCs. Furthermore, inhibition of the proteins regulating resection in G1 phase, like PLK3, not only diminished the formation of pRPA foci but also impaired DSB repair in ESCs. Whereas, in non-pluripotent cells (NSCs or HeLa cells), no repair defect was observed after inhibition of resection in G1 phase. These observations revealed that ESCs perform more resection than NSCs, suggesting that resection dependent-NHEJ is a prominent DSB repair pathways in G1-phase ESCs. All together, the data implies that, ESCs can perform long-range resection of DSB ends and, therefore, in case of impaired classical repair pathways, can readily switch to PARP1-dependent alt-NHEJ. Previously, it was shown that nascent RNAs mediate an error-free c-NHEJ by serving as templates to faithfully restore the lost genomic information at the break site. Moreover, it was demonstrated that RNA transcription machinery functions as a molecular motor to promote excessive DNA resection. These evidences led to the curiosity to understand if RNA mediates the long range resection in G1-phase ESCs. The detection of RNA-DNA hybrids at the DSB sites, as well as, reduction in pRPA foci level after inhibition of transcription verified our hypothesis that RNA transcription machinery might mediate long-range resection in G1 phase ESCs. Furthermore, destabilization of the RNA-DNA hybrids by overexpression of RNaseH1 enzyme, induced a significant repair defect in G1 phase ESCs. This effect was identical to the repair defect which was observed after inhibition of PLK3. These observations confirmed the role of RNA in mediating resection in G1-phase ESCs. These findings suggest that, the involvement of RNA as a template during DSB repair might be mediating an error-free repair and thus play an important role in maintaining the genomic integrity of pluripotent stem cells. |
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Alternatives oder übersetztes Abstract: |
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URN: | urn:nbn:de:tuda-tuprints-69751 | ||||
Sachgruppe der Dewey Dezimalklassifikatin (DDC): | 500 Naturwissenschaften und Mathematik > 570 Biowissenschaften, Biologie | ||||
Fachbereich(e)/-gebiet(e): | 10 Fachbereich Biologie > Radiation Biology and DNA Repair 10 Fachbereich Biologie |
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Hinterlegungsdatum: | 17 Dez 2017 20:56 | ||||
Letzte Änderung: | 17 Dez 2017 20:56 | ||||
PPN: | |||||
Referenten: | Löbrich, Prof. Dr. Markus ; Nuber, Prof. Dr. Ulrike ; Laube, Prof. Dr. Bodo ; Rödel, Prof. Dr. Franz | ||||
Datum der mündlichen Prüfung / Verteidigung / mdl. Prüfung: | 14 November 2017 | ||||
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