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The Contribution of PARP-1 and ELG1/ATAD5 to Genomic Stability


Weller, Marie-Christine. The Contribution of PARP-1 and ELG1/ATAD5 to Genomic Stability. 2016, University of Zurich, Faculty of Science.

Abstract

DNA damage is a common threat to all cells, as it is a driver of malignant transformation. However, it can also be exploited in cancer therapy. Commonly used chemotherapeutics induce a high load of DNA lesions, which overwhelm the repair capacity of replicating tumor cells. Novel approaches aim at targeting backup DNA repair pathways in order to induce synthetic lethality in cancer cells that display specific defects in certain DNA repair enzymes. PARP inhibitors, such as Olaparib, are the prime example and have proven to be highly effective in the treatment of BRCA1- and BRCA2-deficient tumors. PARP1 is an enzyme involved in the repair of DNA single- strand breaks (SSBs). These small lesions may collapse into deleterious double-strand breaks (DSBs) when they collide with the replication machinery. In normal cells, DSBs are repaired in an accurate manner by the homologous recombination (HR) repair pathway, which relies on the activity of functional BRCA proteins. HR-deficient tumor cells die when treated with PARP inhibitors due to an accumulation of cytotoxic DSBs, which are then channeled into alternative, error-prone pathways, leading to genome rearrangements and ultimately apoptosis. PARP inhibitors are used as single-agent therapies, suggesting that the origin of lesions that rely on PARP activity must be endogenous. Intracellular reactive oxidative species (ROS) can lead to an accumulation of 8-oxo-2'-deoxyguanosine (GO) in genomic DNA, which is potentially mutagenic due to its base-pairing properties with both cytosine (C) as well as adenine (A). GO-containing lesions are repaired by the base-excision repair (BER) pathway, initiated by two different glycosylases: MYH addresses GO:A pairs, removing the misincorporated A, while OGG1 excises the oxidized guanine (GO) directly, but only from GO:C base-pairs. Transient SSBs are generated during BER, which likely activate PARP that primes them for repair. Due to the abundance of endogenous oxidative DNA damage, we reasoned that their repair by BER leads to the generation of transient SSBs, which are the main contributors to the efficacy of PARP inhibitors in cells lacking active HR. Indeed, we show that MYH-depletion attenuates the sensitivity and genomic instability induced by Olaparib in HR-deficient cells. These results prove that processing of oxidative DNA lesions contributes to PARP inhibitor toxicity and therefore imply that tissue oxygenation and MYH status affect the efficacy of treatment.
An additional study in this thesis focused on another putative DNA repair factor, ATAD5, which has been proposed to be a suppressor of genome instability. Also we observed a hypersensitivity of ATAD5-deficient cells to certain DNA damaging drugs, such as the methylating agent MNNG, the interstrand crosslinking agent MMC and the PARP inhibitor Olaparib. Interestingly, ATAD5-deficiency causes retention of PCNA and ubiquitylated PCNA on chromatin, suggesting that ATAD5 is involved in their unloading from DNA. However, it is unclear at the moment if, and how, PCNA retention on chromatin leads to genome instability. The interaction with PCNA further suggests a role for ATAD5 in DNA replication, but we show here that this is in fact not the case. Instead, it might be required for post-replicative repair or other DNA repair processes, which remains to be investigated in the future. We have generated a useful set of tools to study the contribution of PCNA modifications on the genomic instability induced by ATAD5-deficiency, which will hopefully shed light onto important functions of both ATAD5 and PCNA in DNA metabolism.

Abstract

DNA damage is a common threat to all cells, as it is a driver of malignant transformation. However, it can also be exploited in cancer therapy. Commonly used chemotherapeutics induce a high load of DNA lesions, which overwhelm the repair capacity of replicating tumor cells. Novel approaches aim at targeting backup DNA repair pathways in order to induce synthetic lethality in cancer cells that display specific defects in certain DNA repair enzymes. PARP inhibitors, such as Olaparib, are the prime example and have proven to be highly effective in the treatment of BRCA1- and BRCA2-deficient tumors. PARP1 is an enzyme involved in the repair of DNA single- strand breaks (SSBs). These small lesions may collapse into deleterious double-strand breaks (DSBs) when they collide with the replication machinery. In normal cells, DSBs are repaired in an accurate manner by the homologous recombination (HR) repair pathway, which relies on the activity of functional BRCA proteins. HR-deficient tumor cells die when treated with PARP inhibitors due to an accumulation of cytotoxic DSBs, which are then channeled into alternative, error-prone pathways, leading to genome rearrangements and ultimately apoptosis. PARP inhibitors are used as single-agent therapies, suggesting that the origin of lesions that rely on PARP activity must be endogenous. Intracellular reactive oxidative species (ROS) can lead to an accumulation of 8-oxo-2'-deoxyguanosine (GO) in genomic DNA, which is potentially mutagenic due to its base-pairing properties with both cytosine (C) as well as adenine (A). GO-containing lesions are repaired by the base-excision repair (BER) pathway, initiated by two different glycosylases: MYH addresses GO:A pairs, removing the misincorporated A, while OGG1 excises the oxidized guanine (GO) directly, but only from GO:C base-pairs. Transient SSBs are generated during BER, which likely activate PARP that primes them for repair. Due to the abundance of endogenous oxidative DNA damage, we reasoned that their repair by BER leads to the generation of transient SSBs, which are the main contributors to the efficacy of PARP inhibitors in cells lacking active HR. Indeed, we show that MYH-depletion attenuates the sensitivity and genomic instability induced by Olaparib in HR-deficient cells. These results prove that processing of oxidative DNA lesions contributes to PARP inhibitor toxicity and therefore imply that tissue oxygenation and MYH status affect the efficacy of treatment.
An additional study in this thesis focused on another putative DNA repair factor, ATAD5, which has been proposed to be a suppressor of genome instability. Also we observed a hypersensitivity of ATAD5-deficient cells to certain DNA damaging drugs, such as the methylating agent MNNG, the interstrand crosslinking agent MMC and the PARP inhibitor Olaparib. Interestingly, ATAD5-deficiency causes retention of PCNA and ubiquitylated PCNA on chromatin, suggesting that ATAD5 is involved in their unloading from DNA. However, it is unclear at the moment if, and how, PCNA retention on chromatin leads to genome instability. The interaction with PCNA further suggests a role for ATAD5 in DNA replication, but we show here that this is in fact not the case. Instead, it might be required for post-replicative repair or other DNA repair processes, which remains to be investigated in the future. We have generated a useful set of tools to study the contribution of PCNA modifications on the genomic instability induced by ATAD5-deficiency, which will hopefully shed light onto important functions of both ATAD5 and PCNA in DNA metabolism.

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Additional indexing

Item Type:Dissertation
Referees:Jiricny Josef, Matthias Peter, Heinz Jacobs
Communities & Collections:04 Faculty of Medicine > Institute of Molecular Cancer Research
07 Faculty of Science > Institute of Molecular Cancer Research
Dewey Decimal Classification:570 Life sciences; biology
610 Medicine & health
Language:English
Date:2016
Deposited On:14 Sep 2016 14:42
Last Modified:31 Aug 2017 07:09

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