Waizenegger, Anja (2016)
Investigation of factors involved in late stages of homologous recombination.
Technische Universität Darmstadt
Dissertation, Erstveröffentlichung
Kurzbeschreibung (Abstract)
Homologous recombination (HR) is an important mechanism to maintain genomic stability, as it is involved in the repair of double-strand breaks (DSBs) and the stabilization of replication forks. To initiate HR, the DNA ends at the break site are resected, generating single-stranded DNA (ssDNA) which is then covered by Rad51 molecules and represents the nucleoprotein filament. During a process called synapsis, this nucleoprotein filament performs homology search and strand invasion within the undamaged sister chromatid. To allow recombination-associated DNA synthesis, using the homologous DNA sequence as a template for repair, Rad51 molecules need to be removed from the DNA. This removal is promoted by the motor protein Rad54, which actively translocates along the heteroduplex DNA and permits subsequent binding of the polymerase to finalize HR. The enzymatic steps and proteins involved in early HR are well established, but later steps of recombination are yet less defined. In this study two new factors, Nek1 and ATRX, were identified which are involved in these late steps of HR, thereby contributing to a better understanding of this important repair pathway. In the publication “Nek1 Regulates Rad54 to Orchestrate Homologous Recombination and Replication Fork Stability”, the never in mitosis A related kinase 1 (Nek1) was shown to regulate the function of Rad54 by phosphorylation at serine 572. This phosphorylation allows removal of Rad51 in late G2 phase, which is necessary to proceed with subsequent HR steps. In S phase however, Rad54 is not phosphorylated and thereby not enabled to remove Rad51 from replication forks, as Rad51 functions in the protection of stalled replication forks from nucleolytic degradation. Nek1 depletion or expression of an unphosphorylatable form of Rad54 (Rad54-S572A) resulted in a G2-specific HR repair defect due to persisting Rad51 molecules on the DNA. In contrast, expression of a phosphomimic form of Rad54 (Rad54-S572E) did not impair HR in G2, but promoted undesired removal of Rad51 from stalled replication forks in S phase. Therefore, Nek1 is a crucial factor for the regulation of Rad54 during the cell cycle, contributing to replication fork stability in S phase and HR functionality in G2 phase. Another novel HR factor was identified in the manuscript “Involvement of ATRX in Homologous Recombination Repair”. Alpha-thalassemia mental retardation X-linked protein (ATRX) was shown here to function during post-synaptic steps of HR. Depletion of ATRX resulted in reduced HR-frequencies in reporter assays, increased numbers of unrepaired DSBs in G2 at late times after irradiation (IR), and the absence of IR-induced sister chromatin exchanges (SCEs). The formation of Rad51 foci as a readout for ongoing HR repair was not affected in ATRX depleted cells compared to control cells and also the removal at late times was almost normal. This indicated an involvement of ATRX downstream of Rad54-dependent Rad51 removal. Furthermore we could show that ATRX interacts with the DNA clamp protein PCNA (proliferating cell nuclear antigen), which is known to be involved in the subsequent step of DNA-synthesis. Strikingly, we identified a PIP box within the ATRX amino acid sequence, which potentially mediates the interaction with PCNA. Therefore, we discovered a function for ATRX besides its established role during replication in S phase and could establish, that ATRX functions in G2-phase during HR, where it interacts with PCNA.
Typ des Eintrags: | Dissertation | ||||
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Erschienen: | 2016 | ||||
Autor(en): | Waizenegger, Anja | ||||
Art des Eintrags: | Erstveröffentlichung | ||||
Titel: | Investigation of factors involved in late stages of homologous recombination | ||||
Sprache: | Englisch | ||||
Referenten: | Layer, Prof. Paul ; Cardoso, Prof. Cristina | ||||
Publikationsjahr: | 8 September 2016 | ||||
Ort: | Darmstadt | ||||
Datum der mündlichen Prüfung: | 4 August 2016 | ||||
URL / URN: | http://tuprints.ulb.tu-darmstadt.de/5659 | ||||
Kurzbeschreibung (Abstract): | Homologous recombination (HR) is an important mechanism to maintain genomic stability, as it is involved in the repair of double-strand breaks (DSBs) and the stabilization of replication forks. To initiate HR, the DNA ends at the break site are resected, generating single-stranded DNA (ssDNA) which is then covered by Rad51 molecules and represents the nucleoprotein filament. During a process called synapsis, this nucleoprotein filament performs homology search and strand invasion within the undamaged sister chromatid. To allow recombination-associated DNA synthesis, using the homologous DNA sequence as a template for repair, Rad51 molecules need to be removed from the DNA. This removal is promoted by the motor protein Rad54, which actively translocates along the heteroduplex DNA and permits subsequent binding of the polymerase to finalize HR. The enzymatic steps and proteins involved in early HR are well established, but later steps of recombination are yet less defined. In this study two new factors, Nek1 and ATRX, were identified which are involved in these late steps of HR, thereby contributing to a better understanding of this important repair pathway. In the publication “Nek1 Regulates Rad54 to Orchestrate Homologous Recombination and Replication Fork Stability”, the never in mitosis A related kinase 1 (Nek1) was shown to regulate the function of Rad54 by phosphorylation at serine 572. This phosphorylation allows removal of Rad51 in late G2 phase, which is necessary to proceed with subsequent HR steps. In S phase however, Rad54 is not phosphorylated and thereby not enabled to remove Rad51 from replication forks, as Rad51 functions in the protection of stalled replication forks from nucleolytic degradation. Nek1 depletion or expression of an unphosphorylatable form of Rad54 (Rad54-S572A) resulted in a G2-specific HR repair defect due to persisting Rad51 molecules on the DNA. In contrast, expression of a phosphomimic form of Rad54 (Rad54-S572E) did not impair HR in G2, but promoted undesired removal of Rad51 from stalled replication forks in S phase. Therefore, Nek1 is a crucial factor for the regulation of Rad54 during the cell cycle, contributing to replication fork stability in S phase and HR functionality in G2 phase. Another novel HR factor was identified in the manuscript “Involvement of ATRX in Homologous Recombination Repair”. Alpha-thalassemia mental retardation X-linked protein (ATRX) was shown here to function during post-synaptic steps of HR. Depletion of ATRX resulted in reduced HR-frequencies in reporter assays, increased numbers of unrepaired DSBs in G2 at late times after irradiation (IR), and the absence of IR-induced sister chromatin exchanges (SCEs). The formation of Rad51 foci as a readout for ongoing HR repair was not affected in ATRX depleted cells compared to control cells and also the removal at late times was almost normal. This indicated an involvement of ATRX downstream of Rad54-dependent Rad51 removal. Furthermore we could show that ATRX interacts with the DNA clamp protein PCNA (proliferating cell nuclear antigen), which is known to be involved in the subsequent step of DNA-synthesis. Strikingly, we identified a PIP box within the ATRX amino acid sequence, which potentially mediates the interaction with PCNA. Therefore, we discovered a function for ATRX besides its established role during replication in S phase and could establish, that ATRX functions in G2-phase during HR, where it interacts with PCNA. |
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URN: | urn:nbn:de:tuda-tuprints-56598 | ||||
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 |
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Hinterlegungsdatum: | 04 Dez 2016 20:55 | ||||
Letzte Änderung: | 04 Dez 2016 20:55 | ||||
PPN: | |||||
Referenten: | Layer, Prof. Paul ; Cardoso, Prof. Cristina | ||||
Datum der mündlichen Prüfung / Verteidigung / mdl. Prüfung: | 4 August 2016 | ||||
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