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Investigations to the DNA resection regulation of ion-induced DSBs in G1 cells and the resection limitations of the cells in quiescent state

Syzonenko, Tatyana (2018):
Investigations to the DNA resection regulation of ion-induced DSBs in G1 cells and the resection limitations of the cells in quiescent state.
Darmstadt, Technische Universität, [Online-Edition: http://tuprints.ulb.tu-darmstadt.de/7456],
[Ph.D. Thesis]

Abstract

Complex DNA double-strand breaks (DSB) are the most severe DNA damage in that they represent an enormous challenge for the cell to repair faithfully as well as repairing at all. Two main repair pathways are known to repair DSBs: classical non-homologous end joining (c-NHEJ) and homologous recombination (HR). c-NHEJ is available in all cell cycle phases whereas the HR pathway functions only in S/G2 when the sister chromatid is available as a template. In addition, a further repair pathway is available to the cells – alternative non-homologous end joining (alt- NHEJ), which can operate on resected DNA break ends. DNA resection is an important step in the cell’s decision as to which DSB repair pathway to choose. DNA resection takes place not only in S/G2 cells but also in G1 cells following complex DNA damage induced by heavy ion radiation. The goal of this thesis is to elucidate the mechanism of resection regulation in G1 cells after heavy ion irradiation. It specifically focuses on the role of the resection regulatory factors RIF1 and BRCA1. A useful model to study the mechanism of resection limitation are cells in quiescent state, G0 cells. In this thesis, quiescent human fibroblasts were used due to their limited resection of heavy ion- induced DSBs in comparison to proliferating human fibroblasts in G1 phase. Therefore, the further aim was to investigate the role of quiescent state compared to the proliferating state in the resection regulation after low- and high LET irradiation. Recruitment of RIF1 to heavy ion induced DSBs in S/G2 and G1 phase cells suggested its involvement in the DNA damage response of complex DNA lesions. RIF1’s recruitment to DSB sites is LET dependent in S/G2 cells. However, a direct impairment of DNA resection of ion- induced DSBs by RIF1 was not detected. An RPA-foci formation analysis upon ion irradiation in BRCA1 depleted cells, however, it showed that the RIF1 antagonist BRCA1 is involved in resection regulation of ion-induced DSBs in G1 cells. Moreover, BRCA1 depletion resulted in a strong increase of the number of RIF1 foci in G1 cells after carbon ion irradiation suggesting that RIF1 is, after all, involved in the resection regulation of heavy ion induced DSBs. This was further supported by the observation that RIF1 depletion led to an increase of the BRCA1 foci number in G1 cells. Co-immunofluorescence staining of BRCA1 and RIF1 showed cell cycle dependent overlap of the BRCA1 and RIF1 foci, which was more pronounced in G1 than in S/G2 cells. The observed overlap of BRCA1 and RPA foci was also cell cycle dependent, yet was greater in S/G2 compared to G1 cells suggesting that BRCA1 is more active in the resection promotion after heavy ion radiation in S/G2 than in G1 cells. UHRF1 is as an interaction partner of BRCA1 in removing RIF1 from DSBs. Therefore, UHRF1 was considered as a positive resection regulator and hence was depleted in U2OS cells to analyze its influence on resection of ion-induced DSBs. Surprisingly, UHRF1 depletion strongly decreased the fraction of resection positive G1 and S/G2 cells indicating a significant role of UHRF1 in the resection regulation of complex DSBs. Normal human fibroblasts in G0 phase showed a smaller number of resection positive cells than fibroblasts in G1 phase. To analyze the cause of decreased resection in human fibroblasts a XI protocol to enrich the cells in G1 or G0 phase was established. The biochemical characteristics of human fibroblasts in G1 and G0 phase as well as DSB repair-kinetics after X-ray irradiation showed essential differences between these two cell cycle states. The examination of repair factors that promote resection by Western analysis revealed that BRCA1 and CtIP were strongly reduced in quiescent cells. Moreover, the resection antagonists 53BP1 and Ku80, which protect the DSB ends from resection to promote c-NHEJ, were available. Interestingly, RIF1, which represents another resection antagonist, was not detectable in quiescent cells. The RIF1 downregulation is in line with its recently discovered role in the DNA replication machinery. Furthermore, together with the decreased fraction of resection positive cells after heavy ion irradiation, G0 cells showed slower repair kinetics of X-ray induced DSBs than fibroblasts in G1 phase. A 24 h post irradiation G0 cells revealed still 20 - 30 % unrepaired DSBs detected by gH2AX immunofluorescence staining whereas DSBs in G1 cells were virtually all repaired. The possibility that G0 and G1 cells utilize different DSB repair pathways was examined by using DNA-repair pathway inhibitors; a DNA-PKcs inhibitor was employed in order to inhibit c-NHEJ and a PARP inhibitor was used to inhibit alt-NHEJ. The severe repair impairment of X-ray induced DSBs by the DNA-PKcs inhibitor and the lack of influence by the PARP inhibitor indicated that the main repair pathway for both G1 and G0 cells is c-NHEJ to repair this type of damage. Interestingly, the repair kinetics of helium ion-induced DSBs in G1 and G0 cells was identical, suggesting that c-NHEJ represents the main repair pathway. Analysis of heterochromatin marker trimethylated histone H3 at amino acids lysine 9 (H3K9Me3) and lysine 27 (H3K27Me3) suggested that G0 cells have more compact chromatin than G1 cell. This may well be the reason why G0 cells show different repair kinetics of X-ray induced DSBs compared to G1 cells, as the chromatin status influences the DSB-repair pathway choice and thus repair kinetics. The comparative analysis by clonogenic survival assay of delayed plated G1 versus G0 cells after ionizing radiation mirrored the repair kinetics after X-ray- and heavy ion irradiation. Post X-ray irradiation G0 cells were more sensitive than G1 cells, whereas after carbon ion irradiation the G0 and G1 cells showed almost the same radiosensitivity.Taken together this thesis showed, that both G1 and G0 cells can efficiently repair heavy ion induced damage by DNA-PKcs dependent repair pathway. However, a different chromatin structure might be a cause of slower repair kinetics in G0 cells after X-ray irradiation compared to G1 cells. A slower repair kinetics after Helium ion irradiation compared to X-ray irradiation in G1 cells suggests that G1 cells use a resection dependent c-NHEJ to repair complex DSBs.

Item Type: Ph.D. Thesis
Erschienen: 2018
Creators: Syzonenko, Tatyana
Title: Investigations to the DNA resection regulation of ion-induced DSBs in G1 cells and the resection limitations of the cells in quiescent state
Language: English
Abstract:

Complex DNA double-strand breaks (DSB) are the most severe DNA damage in that they represent an enormous challenge for the cell to repair faithfully as well as repairing at all. Two main repair pathways are known to repair DSBs: classical non-homologous end joining (c-NHEJ) and homologous recombination (HR). c-NHEJ is available in all cell cycle phases whereas the HR pathway functions only in S/G2 when the sister chromatid is available as a template. In addition, a further repair pathway is available to the cells – alternative non-homologous end joining (alt- NHEJ), which can operate on resected DNA break ends. DNA resection is an important step in the cell’s decision as to which DSB repair pathway to choose. DNA resection takes place not only in S/G2 cells but also in G1 cells following complex DNA damage induced by heavy ion radiation. The goal of this thesis is to elucidate the mechanism of resection regulation in G1 cells after heavy ion irradiation. It specifically focuses on the role of the resection regulatory factors RIF1 and BRCA1. A useful model to study the mechanism of resection limitation are cells in quiescent state, G0 cells. In this thesis, quiescent human fibroblasts were used due to their limited resection of heavy ion- induced DSBs in comparison to proliferating human fibroblasts in G1 phase. Therefore, the further aim was to investigate the role of quiescent state compared to the proliferating state in the resection regulation after low- and high LET irradiation. Recruitment of RIF1 to heavy ion induced DSBs in S/G2 and G1 phase cells suggested its involvement in the DNA damage response of complex DNA lesions. RIF1’s recruitment to DSB sites is LET dependent in S/G2 cells. However, a direct impairment of DNA resection of ion- induced DSBs by RIF1 was not detected. An RPA-foci formation analysis upon ion irradiation in BRCA1 depleted cells, however, it showed that the RIF1 antagonist BRCA1 is involved in resection regulation of ion-induced DSBs in G1 cells. Moreover, BRCA1 depletion resulted in a strong increase of the number of RIF1 foci in G1 cells after carbon ion irradiation suggesting that RIF1 is, after all, involved in the resection regulation of heavy ion induced DSBs. This was further supported by the observation that RIF1 depletion led to an increase of the BRCA1 foci number in G1 cells. Co-immunofluorescence staining of BRCA1 and RIF1 showed cell cycle dependent overlap of the BRCA1 and RIF1 foci, which was more pronounced in G1 than in S/G2 cells. The observed overlap of BRCA1 and RPA foci was also cell cycle dependent, yet was greater in S/G2 compared to G1 cells suggesting that BRCA1 is more active in the resection promotion after heavy ion radiation in S/G2 than in G1 cells. UHRF1 is as an interaction partner of BRCA1 in removing RIF1 from DSBs. Therefore, UHRF1 was considered as a positive resection regulator and hence was depleted in U2OS cells to analyze its influence on resection of ion-induced DSBs. Surprisingly, UHRF1 depletion strongly decreased the fraction of resection positive G1 and S/G2 cells indicating a significant role of UHRF1 in the resection regulation of complex DSBs. Normal human fibroblasts in G0 phase showed a smaller number of resection positive cells than fibroblasts in G1 phase. To analyze the cause of decreased resection in human fibroblasts a XI protocol to enrich the cells in G1 or G0 phase was established. The biochemical characteristics of human fibroblasts in G1 and G0 phase as well as DSB repair-kinetics after X-ray irradiation showed essential differences between these two cell cycle states. The examination of repair factors that promote resection by Western analysis revealed that BRCA1 and CtIP were strongly reduced in quiescent cells. Moreover, the resection antagonists 53BP1 and Ku80, which protect the DSB ends from resection to promote c-NHEJ, were available. Interestingly, RIF1, which represents another resection antagonist, was not detectable in quiescent cells. The RIF1 downregulation is in line with its recently discovered role in the DNA replication machinery. Furthermore, together with the decreased fraction of resection positive cells after heavy ion irradiation, G0 cells showed slower repair kinetics of X-ray induced DSBs than fibroblasts in G1 phase. A 24 h post irradiation G0 cells revealed still 20 - 30 % unrepaired DSBs detected by gH2AX immunofluorescence staining whereas DSBs in G1 cells were virtually all repaired. The possibility that G0 and G1 cells utilize different DSB repair pathways was examined by using DNA-repair pathway inhibitors; a DNA-PKcs inhibitor was employed in order to inhibit c-NHEJ and a PARP inhibitor was used to inhibit alt-NHEJ. The severe repair impairment of X-ray induced DSBs by the DNA-PKcs inhibitor and the lack of influence by the PARP inhibitor indicated that the main repair pathway for both G1 and G0 cells is c-NHEJ to repair this type of damage. Interestingly, the repair kinetics of helium ion-induced DSBs in G1 and G0 cells was identical, suggesting that c-NHEJ represents the main repair pathway. Analysis of heterochromatin marker trimethylated histone H3 at amino acids lysine 9 (H3K9Me3) and lysine 27 (H3K27Me3) suggested that G0 cells have more compact chromatin than G1 cell. This may well be the reason why G0 cells show different repair kinetics of X-ray induced DSBs compared to G1 cells, as the chromatin status influences the DSB-repair pathway choice and thus repair kinetics. The comparative analysis by clonogenic survival assay of delayed plated G1 versus G0 cells after ionizing radiation mirrored the repair kinetics after X-ray- and heavy ion irradiation. Post X-ray irradiation G0 cells were more sensitive than G1 cells, whereas after carbon ion irradiation the G0 and G1 cells showed almost the same radiosensitivity.Taken together this thesis showed, that both G1 and G0 cells can efficiently repair heavy ion induced damage by DNA-PKcs dependent repair pathway. However, a different chromatin structure might be a cause of slower repair kinetics in G0 cells after X-ray irradiation compared to G1 cells. A slower repair kinetics after Helium ion irradiation compared to X-ray irradiation in G1 cells suggests that G1 cells use a resection dependent c-NHEJ to repair complex DSBs.

Place of Publication: Darmstadt
Divisions: 10 Department of Biology
10 Department of Biology > Radiation Biology and DNA Repair
Date Deposited: 03 Jun 2018 19:55
Official URL: http://tuprints.ulb.tu-darmstadt.de/7456
URN: urn:nbn:de:tuda-tuprints-74567
Referees: Taucher-Scholz, Prof.Dr. Gisela and Durante, Prof.Dr. Marco
Refereed / Verteidigung / mdl. Prüfung: 23 January 2018
Alternative Abstract:
Alternative abstract Language
Komplexe DNA Doppelstrangbrüche (DSB) zählen zu den schwerwiegendsten DNA Schäden. Die Zellen können die DSB über zwei Hauptreparaturwege: die klassische nicht-homologe End- Verknüpfung (c-NHEJ, engl. classic non-homologous end joining) und die homologe Rekombination (HR) reparieren. C-NHEJ ist in allen Zellzyklus-Phasen verfügbar, während die homologe Rekombination nur in S/G2-Zellen zur Verfügung steht, wo die Schwesterchromatiden als Vorlagen dienen. Des Weiteren besitzen die Zellen einen alternativen DSB-Reparaturweg – die alternative nicht homologe End-Verknüpfung (alt-NHEJ, engl. alternative non-homologous end joining). Dieser Reparaturweg kann auch resektierte DSB-Enden verknüpfen. Durch die initiale DSB-Resektion wird über die Auswahl des Reparaturweges entschieden. Aktuelle Studien zeigen, dass die DSB-Resektion nicht nur in S/G2- sondern auch in G1-Zellen nach Schwerionenbestrahlung auftritt. Das Ziel dieser Arbeit ist den Mechanismus der Resektionsregulation an den DSB nach Schwerionenbestrahlung in G1-Zellen weiter aufzuklären. Dabei stand die Rolle der Resektionsfaktoren BRCA1 und RIF1 im Vordergrund. Es konnte gezeigt werden, dass RIF1 zu schwerioneninduzierten DSB rekrutiert wird, was auf eine Beteiligung an komplexen DNA Schäden hinweisen könnte. Außerdem zeigte sich eine LET- Abhängigkeit der RIF1-Rekrutierung an DSB in S/G2-Zellen. Ein direkter Einfluss von RIF1 auf Resektion nach Herunterregulierung der RIF1-Expression durch RNAi war jedoch nicht nachweisbar. Eine RNAi-basierter Knockdown der BRCA1-Expression hingegen reduzierte Resektion in S/G2- und G1-Zellen. Eine erhöhte Anzahl an RIF1-Foci in BRCA1-Knockdown- Zellen deutete indirekt auf einen Einfluss von RIF1 auf DSB-Resektion nach Kohlenstoffbestrahlung hin. Die antagonistische Wirkung von BRCA1 und RIF1 zeigte sich weiterhin im Anstieg der BRCA1-Foci nach Kohlenstoffbestrahlung in G1- und S/G2-Zellen, deren RIF1-Expression durch RNAi herunterreguliert war. Kolokalisationsanalysen in Immunfluoreszenzfärbungen mit BRCA1 und RIF1 zeigten eine Überlagerung der Signale, wobei die Ausprägung dieser Überlagerung in G1- stärker als in S/G2- Zellen war. RPA und BRCA1 zeigten ebenfalls eine Überlagerung der Fluoreszenzsignale, wobei hier die Ausprägung der Überlagerung in S/G2- größer als in G1-Zellen war. Dieses könnte ein Hinweis darauf sein, dass BRCA1 aktiver in der Resektionsregulation in S/G2- als in G1-Zellen ist. UHRF1 ist ein BRCA1-Interaktionspartner und agiert an den DSB und ist verantwortlich dafür RIF1 vom Schaden zu entfernen. In dieser Arbeit konnte gezeigt werden, dass UHRF1- Knockdown sowohl die Fraktion resektionspositiver S/G2- als auch G1 Zellen nach Schwerionenbestrahlung reduzierte. Dieses Ergebnis zeigt, dass UHRF1 in der Resektions- regulation an den komplexen DSB beteiligt ist. Es wurde ein kleinerer Anteil der resektionspositiven Zellen nach Schwerionenbestrahlung in humanen Fibroblasten in G0-Phase im Vergleich zu G1-Phase gezeigt. Die Untersuchungen der Reparaturfaktoren und der Reparaturkinetik der DSB nach Röntgenbestrahlung zeigen deutliche Unterschiede zwischen humanen Fibroblasten in G0- und in G1-Phase. Die positiven XIII Resektionsfaktoren BRCA1 und CtIP waren in G0-Zellen kaum vorhanden. Die Resektionsantagonisten 53BP1 und Ku80, welche die DSB-Enden gegen Resektion schützen, waren vorhanden. Hingegen war der resektionsantagonistische Faktor RIF1 in G0-Zellen nicht nachweisbar. Neben der verminderten Resektion zeigten die G0-Zellen eine verlangsamte Reparatur-kinetik im Vergleich zu G1-Zellen. 24 Stunden nach der Röntgenbestrahlung zeigten G0 Zellen in einer gH2AX- Immunfluoreszenzfärbung immer noch 20 - 30 % nicht-reparierte DSB, während die DSB in G1-Zellen fast vollständig repariert wurden. Die Frage, ob dieser Unterschied auf der Verwendung unterschiedlicher Reparaturwege in G0- bzw. G1-Zellen beruht, wurde mit geeigneten Inhibitoren gegen c-NHEJ bzw. alt-NHEJ überprüft. C-NHEJ-Inhibition, aber nicht alt- NHEJ-Inhibition, beeinträchtigte die DSB Reparatur deutlich. Den Hauptreparaturweg nach Röntgenbestrahlung sowohl in G0 als auch in G1-Zellen stellt daher der c-NHEJ dar. Im Gegensatz dazu, war die Reparaturkinetik nach der Heliumbestrahlung in G1- und G0-Zellen identisch. Der Hauptreparaturweg war ebenfalls c-NHEJ. Die Charakterisierung der Chromatinverdichtung mit den Heterochromatinmarkern trimethyliertes Histon H3 an der Aminosäuren Lysin 9 (H3K9Me3) und Lysin 27 (H3K27Me3) deutete darauf hin, dass G0-Zellen eine höhere Chromatinkompaktierung als G1-Zellen aufweisen. Da der Chromatinzustand die Auswahl des DSB-Reparaturweges und somit auch die Reparaturkinetik beeinflusst, kann der Unterschied in der Chromatinkompaktierung zwischen G0- und G1-Zellen ein Grund für die unterschiedliche Reparaturkinetik sein. Um die Qualität der DSB-Reparatur zu untersuchen, wurden die G1- und G0-Zellen in einem Zellüberlebensexperiment quantitativ analysiert. Die so erzielten Ergebnisse sind im Einklang mit der Reparaturkinetik nach Röntgen- und Schwerionenbestrahlung. Dabei hatten G0-Zellen 24 Stunden nach der Röntgenbestrahlung eine höhere Strahlenempfindlichkeit als G1-Zellen, während die Strahlenempfindlichkeit nach Kohlenstoffbestrahlung bei beiden vergleichbar war. Zusammenfassend konnte es in dieser Arbeit gezeigt werden, dass die G0- und G1-Zellen eine effiziente Reparatur nach Schwerionenstrahlung durchführen können, wobei beide einen DNA- PKcs-abhängigen Reparaturweg benutzen. Hingegen, könnte der Unterschied in der Chromatinstruktur ein Grund für die verlangsamte Reparaturkinetik nach Röntgenbestrahlung in G0-Zellen im Vergleich zu G1-Zellen sein. Die langsame Reparaturkinetik nach Heliumionenbestrahlung könnte im Vergleich zu Röntgenbestrahlung in G1-Zellen am resektionsabhängigen c-NHEJ liegen.German
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