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Characterization of Rad52 protein function during DNA double-strand break repair in G2 phase mammalian cells

Gawai, Anugrah (2018)
Characterization of Rad52 protein function during DNA double-strand break repair in G2 phase mammalian cells.
Technische Universität Darmstadt
Dissertation, Erstveröffentlichung

Kurzbeschreibung (Abstract)

DNA double-strand breaks (DSBs) are the most deleterious damage which cells can encounter. Unrepaired or mis-repaired DSBs can result in genomic instability and cell death. Therefore, DSBs pose a serious threat to genome integrity. Two main repair pathways, canonical non-homologous end-joining (c-NHEJ) and homologous recombination (HR), are known to play a primary role in the repair of DSBs. Cell cycle-specific studies have revealed that c-NHEJ is active throughout the cell cycle, whereas, HR is active in the late-S and G2 phase where a sister chromatid is available as a template for repair. Molecular characterization of these pathways has discovered various key players, such as Ku70/80 and DNA-PKcs of c-NHEJ and Rad51, BRCA2 and Rad54 of HR. Recent findings have made it evident that when c-NHEJ or HR is impaired, alternative end-joining (alt-EJ) pathway operates to remove the DSBs. While alt-EJ acts as a global rescuing mechanism, the removal of DSBs by alt-EJ comes at a cost of elevated chromosomal translocations and sequence alteration at the break ends. Factors implicated in alt-EJ are Mre11, CtIP, DNA ligase 1/3 and PARP1. Recently, DNA polymerase theta (Polθ) was shown to promote alt-EJ by annealing the micro-homologies (MHs), present internal to the resected break ends. Therefore, this pathway is also referred to as DNA polymerase theta-mediated end-joining (TMEJ).

Most of the knowledge about the mechanistic details and the factors involved in HR comes from the studies performed with yeast (S. cerevisiase). In S. cerevisiase, Rad52 was discovered as the key HR player whose absence results in defects in DNA repair, increased sensitivity to IR and cell death. Surprisingly, loss of Rad52 in vertebrate cells has no effect on DNA repair and Rad52 knock-out mice are fertile and viable. However, increasing evidences suggest that Rad52 is involved in HR in mammalian cells. Earlier work performed in the laboratory of Prof. Löbrich showed that Rad52-GFP foci peak in the late G2 phase and persist in the consequent M phase. A study published by Feng et al. (2011) showed that, in BRCA2-deficient mammalian cells, inactivation of Rad52 is synthetically lethal for the cells. However, the exact function of Rad52 in BRCA2-proficient as well as deficient cells is not yet clearly understood.

In this study the function of Rad52 during DSB repair in G2 phase mammalian cells was characterized. In contrast to earlier speculations, in HeLa cells it was shown that Rad52 is not involved in the loading of Rad51 on to the resected 3'-ssDNA overhangs in G2 phase cells and, thus, cannot compensate for the loss of BRCA2. A synthetically lethal relationship was observed between BRCA2 and Rad52, indicating an important role for Rad52 in BRCA2-depleted HeLa and 82-6 (fibroblast) cells. Importantly, by using HeLa-Rad52-GFP cells, it was shown for the first time that BRCA2-depleted cells show significantly increased amounts of Rad52-GFP foci in G2 phase cells. yH2AX foci analysis, however, showed that the increased numbers of Rad52-GFP foci do not imply activation of a Rad52-dependent alternative repair pathway. Interestingly, co-depletion of BRCA2 and Rad52 rescued the BRCA2 repair defect. Furthermore, it was observed that the rescue of the BRCA2 repair defect was due to the activation of the Polθ-mediated TMEJ repair pathway, which gave rise to increased numbers of chromosomal fusions. These results were true for both Hela and fibroblast (82-6 & HSC-62) cells.

Notably, in HeLa cells, it was shown that the two resection-dependent pathways, HR & TMEJ, are active simultaneously in G2 phase. Nevertheless, rescue of the BRCA2-repair defect after depleting Rad52 was still observed in HeLa cells.

In conclusion, the results suggest that due to its ss-DNA binding activity, Rad52 binds to the resected 3'-ssDNA overhangs in BRCA2-deficient cells. This binding of Rad52 prevents TMEJ from repairing the resected DSB ends as repair by TMEJ can result in increased chromosomal fusions. Thus, by preventing the action of TMEJ pathway, Rad52 suppresses the formation of chromosomal fusions and maintains genomic stability in BRCA2-deficient cells. In context to cancer therapy, inactivation of Rad52 in BRCA2-deficient tumors can prove to be a potential therapeutic strategy. Furthermore, in this study it was shown that inactivation of Polθ and BRCA2 results in significantly increased numbers of chromosomal fusions in HeLa cells. This result is consistent with earlier published data where it was shown that inactivation of Polθ in BRCA2-deficient tumors increases chromosomal aberrations and enhances cell killing. Therefore, combined inactivation of Rad52 and Polθ might prove to be more potent and, thus, provide an alternative strategy to specifically kill BRCA2-deficient cancer cells.

Typ des Eintrags: Dissertation
Erschienen: 2018
Autor(en): Gawai, Anugrah
Art des Eintrags: Erstveröffentlichung
Titel: Characterization of Rad52 protein function during DNA double-strand break repair in G2 phase mammalian cells
Sprache: Englisch
Referenten: Löbrich, Prof. Dr. Markus ; Cardoso, Prof. Dr. Cristina M.
Publikationsjahr: Januar 2018
Ort: Darmstadt
Datum der mündlichen Prüfung: 20 Dezember 2017
URL / URN: http://tuprints.ulb.tu-darmstadt.de/7220
Kurzbeschreibung (Abstract):

DNA double-strand breaks (DSBs) are the most deleterious damage which cells can encounter. Unrepaired or mis-repaired DSBs can result in genomic instability and cell death. Therefore, DSBs pose a serious threat to genome integrity. Two main repair pathways, canonical non-homologous end-joining (c-NHEJ) and homologous recombination (HR), are known to play a primary role in the repair of DSBs. Cell cycle-specific studies have revealed that c-NHEJ is active throughout the cell cycle, whereas, HR is active in the late-S and G2 phase where a sister chromatid is available as a template for repair. Molecular characterization of these pathways has discovered various key players, such as Ku70/80 and DNA-PKcs of c-NHEJ and Rad51, BRCA2 and Rad54 of HR. Recent findings have made it evident that when c-NHEJ or HR is impaired, alternative end-joining (alt-EJ) pathway operates to remove the DSBs. While alt-EJ acts as a global rescuing mechanism, the removal of DSBs by alt-EJ comes at a cost of elevated chromosomal translocations and sequence alteration at the break ends. Factors implicated in alt-EJ are Mre11, CtIP, DNA ligase 1/3 and PARP1. Recently, DNA polymerase theta (Polθ) was shown to promote alt-EJ by annealing the micro-homologies (MHs), present internal to the resected break ends. Therefore, this pathway is also referred to as DNA polymerase theta-mediated end-joining (TMEJ).

Most of the knowledge about the mechanistic details and the factors involved in HR comes from the studies performed with yeast (S. cerevisiase). In S. cerevisiase, Rad52 was discovered as the key HR player whose absence results in defects in DNA repair, increased sensitivity to IR and cell death. Surprisingly, loss of Rad52 in vertebrate cells has no effect on DNA repair and Rad52 knock-out mice are fertile and viable. However, increasing evidences suggest that Rad52 is involved in HR in mammalian cells. Earlier work performed in the laboratory of Prof. Löbrich showed that Rad52-GFP foci peak in the late G2 phase and persist in the consequent M phase. A study published by Feng et al. (2011) showed that, in BRCA2-deficient mammalian cells, inactivation of Rad52 is synthetically lethal for the cells. However, the exact function of Rad52 in BRCA2-proficient as well as deficient cells is not yet clearly understood.

In this study the function of Rad52 during DSB repair in G2 phase mammalian cells was characterized. In contrast to earlier speculations, in HeLa cells it was shown that Rad52 is not involved in the loading of Rad51 on to the resected 3'-ssDNA overhangs in G2 phase cells and, thus, cannot compensate for the loss of BRCA2. A synthetically lethal relationship was observed between BRCA2 and Rad52, indicating an important role for Rad52 in BRCA2-depleted HeLa and 82-6 (fibroblast) cells. Importantly, by using HeLa-Rad52-GFP cells, it was shown for the first time that BRCA2-depleted cells show significantly increased amounts of Rad52-GFP foci in G2 phase cells. yH2AX foci analysis, however, showed that the increased numbers of Rad52-GFP foci do not imply activation of a Rad52-dependent alternative repair pathway. Interestingly, co-depletion of BRCA2 and Rad52 rescued the BRCA2 repair defect. Furthermore, it was observed that the rescue of the BRCA2 repair defect was due to the activation of the Polθ-mediated TMEJ repair pathway, which gave rise to increased numbers of chromosomal fusions. These results were true for both Hela and fibroblast (82-6 & HSC-62) cells.

Notably, in HeLa cells, it was shown that the two resection-dependent pathways, HR & TMEJ, are active simultaneously in G2 phase. Nevertheless, rescue of the BRCA2-repair defect after depleting Rad52 was still observed in HeLa cells.

In conclusion, the results suggest that due to its ss-DNA binding activity, Rad52 binds to the resected 3'-ssDNA overhangs in BRCA2-deficient cells. This binding of Rad52 prevents TMEJ from repairing the resected DSB ends as repair by TMEJ can result in increased chromosomal fusions. Thus, by preventing the action of TMEJ pathway, Rad52 suppresses the formation of chromosomal fusions and maintains genomic stability in BRCA2-deficient cells. In context to cancer therapy, inactivation of Rad52 in BRCA2-deficient tumors can prove to be a potential therapeutic strategy. Furthermore, in this study it was shown that inactivation of Polθ and BRCA2 results in significantly increased numbers of chromosomal fusions in HeLa cells. This result is consistent with earlier published data where it was shown that inactivation of Polθ in BRCA2-deficient tumors increases chromosomal aberrations and enhances cell killing. Therefore, combined inactivation of Rad52 and Polθ might prove to be more potent and, thus, provide an alternative strategy to specifically kill BRCA2-deficient cancer cells.

Alternatives oder übersetztes Abstract:
Alternatives AbstractSprache

DNA-Doppelstrangbrüche (DSBs) zählen zu den schwerwiegendsten DNA Schäden, da sie die Integrität des Genoms gefährden. Für die Reparatur von DSBs, stehen zwei Hauptreparaturwege zur Verfügung: die klassiche nicht-homologe Endverknüpfung (c-NHEJ) und die homologe Rekombination (HR). Zellzyklus spezifische Studien haben gezeigt, dass c-NHEJ in allen Zellzyklus phasen aktive ist, wohingegen HR nur in der späten-S und G2 Phase aktive ist wenn ein Schwesterchromatid zur Verfügung steht. Es würden bereits viele Schlüsselfaktoren der beiden Reparaturwege durch molekulare Charakterisierung entdeckt, z.B., Ku70/80 und DNA-PKcs, die am c-NHEJ beteiligt sind und die HR Proteine Rad51, BRCA2 und Rad54.Neue Studien haben erwiesen, dass DSBs in c-NHEJ oder HR-defizienten Zellen, durch alternative Endverknüpfung (alt-EJ) reparierte werden. Jedoch ist Reparatur durch alt-EJ fehleranfällig, und kann zu chromosomalen Translokationen führen sowie Sequenzänderungen an der Bruchstelle. Die Faktoren Mre11, CtIP, DNA ligase 1/3 und PARP1 spielen die Hauptrolle während dem alt-EJ. Vor kurzem Zeit haben Forscher entdeckt, dass DNA polymerase-theta (Polθ) auch im alt-EJ beteiligt ist. Polθ übernimmt hierbei die Aufgabe mikrohomologie Sequenzen, die in resektierten DNA Brüchen versteckt sind, zusammen zu bringen. Daher wird alt-EJ auch theta-vermittelte Endverknüpfung (TMEJ) genannt.

Das meiste Wissen über den Mechanismus der Reparatur durch HR ist von Studien bekannt, die in Hefe (S. cerevisiae) durchgeführt wurden. Rad52 wurde in S. cerevisiae als HR Schlüsselfaktor entdeckt. Die Abwesenheit von Rad52 in S. cerevisiae führt zu Defekten in der DNA Reparatur, erhöhter Strahlensensitivität und zu Zelltod. Erstaunlicherweise, hat die Abwesenheit von Rad52 in Säugerzellen aber keinen Phänotyp gezeigt. In den letzten Jahren gab es Jedoch mehrere Studien, die Hinweise geliefert haben, dass Rad52 eine Rolle bei der HR in Säugerezellen spielt. Frühere Studien der Arbeitsgruppe Löbrich haben gezeigt, nach ionisierender Strahlung (IR) Rad52-GFP Foci in der G2 Phase langsam rekrutiert werden, ein Maximum zu späten Zeitpunkten nach Bestrahlung erreichen und, bis in die M Phase persistieren. Eine Veroffentlichung von Feng et al. (2011) hat außerdem gezeigt, dass die Inaktivierung von Rad52 in BRCA2-defizienten Zellen zu einer synthetischen Lethalität führt. Allerdings, sind die Funktionen von Rad52 in wild type (WT) sowie in BRCA2-defizienten Zellen unbekannt.

In dieser Arbeit wurden die Funktionen von Rad52 in Säugerzellen charakterisiert. Im Gegensatz zu früheren Spekulationen, konnte gezeigt werden, dass Rad52 in HeLa Zellen nicht die Funktion übernimmt Rad51 auf resektierte DNA aufzuladen. Im Rahmen dieser Arbeit konnte zum ersten Mal gezeigt werden, dass in BRCA2-deplietierten Zellen eine signifikant höhere Zahl von Rad52-GFP Foci in der G2 Phase sind. Durch yH2AX Foci analyse wurde verdeutlicht, dass die höhere Zahl an Rad52-GFPFoci allerdings nicht bedeutet dass, ein Rad52-abhängiger alternativer Reparaturweg aktiv ist. Erstaunlicherweise wurde beobachtet, dass eine Doppeldepletion von Rad52 und BRCA2 den BRCA2-Reparaturdefekt aufhebt. Weiterhin, es wurde beobachtet, dass die Reparatur in Rad52 und BRCA2 doppeldepletierten Zellen Polθ-vermitteltes TMEJ darstellt. Es konnte gezeigt werden, dass dieser Reparaturweg zur vermehrtern Entstehung von chromosomalen Fusionen führt. Dieser ergebnisse wurden in HeLa Zellen als auch in humanen Fibroblastzelllinien (82-6 hTert & HSC-62 hTert) gezeigt. In HeLa Zellen es wurde außerdem gezeigt, dass zwei resektions-abhängige Reparaturwege (HR & TMEJ) gleichzeitig in G2 aktiv sind.

Abschließend konnte gezeigt werden, dass in BRCA2-defizienten Zellen Rad52 aufgrund der ss-DNA Bindungsaktivität resektierte DNA Brüche binden kann. Diese bindung verhindert eine Reparatur durch TMEJ und daher auch die Entstehung chromosomalen Fusionen. Daher wird Rad52 für die Erhaltung der genomischen Integrität in BRCA2-defizienten Zellen benötigt. Im Rahmen der Krebs therapie bietet die Inaktivierung von Rad52 daher in BRCA2-defizienten Tumorzellen einen vielversprechenden Therapieansatz. Weiterhin konnte in dieser Arbeit gezeigt werden, dass in HeLa Zellen die Abwesenheit von BRCA2 und Polθ zur Ausbildung von chromosomalen Fusionen führt. Dieses Ergebnis ist im Einklang mit anderen Studien, in denen gezeigt wurde, dass die Inaktivierung von Polθ in BRCA2-defizienten Tumorzellen zum Zelltod führt. Daher konnte die kombinierte Inaktivierung von Rad52 und Polθ in BRCA2-defizienten Tumorzellen sich als eine alternative Strategie in der Krebstherapie erweisen.

Deutsch
URN: urn:nbn:de:tuda-tuprints-72206
Sachgruppe der Dewey Dezimalklassifikatin (DDC): 500 Naturwissenschaften und Mathematik > 570 Biowissenschaften, Biologie
Fachbereich(e)/-gebiet(e): 10 Fachbereich Biologie
10 Fachbereich Biologie > Radiation Biology and DNA Repair
Hinterlegungsdatum: 11 Feb 2018 20:55
Letzte Änderung: 11 Feb 2018 20:55
PPN:
Referenten: Löbrich, Prof. Dr. Markus ; Cardoso, Prof. Dr. Cristina M.
Datum der mündlichen Prüfung / Verteidigung / mdl. Prüfung: 20 Dezember 2017
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