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The role of BRCA2 in the maintenance of genome stability in response to replication stress


Mijic, Sofija. The role of BRCA2 in the maintenance of genome stability in response to replication stress. 2017, University of Zurich, Faculty of Science.

Abstract

A fundamental aspect of living organisms is the accurate replication and maintenance of the genome to ensure the high-fidelity inheritance of genetic information throughout many cell generations. The molecular machinery that replicates the DNA – acting at the so-called replication fork – can be frequently hindered by obstacles of both extracellular and intracellular origin, such as chemicals or UV radiations on one hand, and collision with other processes occurring on the DNA, like gene transcription, on the other. These challenges to DNA replication may lead to transient slowing or stalling of replication forks: we refer to this as replication stress. Cells evolved a variety of mechanisms, which fall under the definition of DNA damage response, that allow them to respond to replication stress. Failure of these mechanisms can lead to DNA damage, and ultimately result in genomic instability, a major driving force of cancer. On the other hand, many chemotherapeutic compounds are designed to induce replication stress, exploiting the stringent requirement of highly replicating cancer cells to continuously replicate their genome. Therefore, investigating the mechanisms underlying replication stress has emerged as a key tool to both understand cancer onset, and to develop new therapeutic approaches.
Our laboratory has recently reported that, upon cellular exposure to genotoxic treatments, replication forks slow down and are frequently remodeled to form a detectable four-way junction at the replication fork, called reversed fork. This transient molecular transaction is considered to be a protective response, required to limit fork breakage in conditions of replication stress. In addition, our laboratory reported that, besides exogenous genotoxic treatments, fork reversal frequently occurs also in response to endogenous molecular processes, which are known to undermine genomic integrity, such as the activation (overexpression or amplification) of cellular proto-oncogenes.
In the current thesis, I will present our efforts to understand what triggers reversed fork formation in different contexts, and what are the factors that contribute to its formation and stability.
In the first part, I describe strategies we designed for the generation of new inducible oncogene overexpression systems in different cellular models, with the aim of describing the common or different molecular consequences downstream of aberrant activation of different oncogenes. This part has led to date to inconclusive results, mainly due to technical difficulties in establishing an efficient and robust oncogene induction system. The importance of this question (oncogene- induced replication stress in cancer) demands additional future work, and fine-tuning of the techniques for a time-controlled oncogene overexpression.
In the second part, I successfully investigated the function in replication fork remodeling of the proteins involved in homologous recombination, with particular attention on the tumor suppressors RAD51 and BRCA2. Besides their established role in double strand break repair via homologous recombination, RAD51 and BRCA2 were in fact known to protect stalled replication forks from extensive nucleolytic degradation. Underlying the importance of this alternative function for these genes, defects in fork protection lead to chromosomal instability, and contribute to the sensitivity of BRCA2-defective tumors to chemotherapeutics by yet-unknown mechanisms. Our results showed that RAD51 contributes to reverse fork formation. Moreover, we found that these structures are progressively degraded in the absence of BRCA2 (loss-of-function mutations in BRCA2 are a common feature of many cancer types). Inhibiting MRE11 nuclease activity or its recruitment to the reversed fork can restore fork integrity and prevent chromosomal breakage. On the contrary, preventing fork degradation by impairing the formation of reversed forks, leads to increased chromosomal breakage in BRCA2-defective cells, being thus detrimental for genome stability. Collectively, our study reveals that fork reversal has a crucial physiological relevance in protecting genome stability upon replication stress, and that a complex interplay of HR factors co- operate to remodel and stabilize stalled DNA replication forks.

Abstract

A fundamental aspect of living organisms is the accurate replication and maintenance of the genome to ensure the high-fidelity inheritance of genetic information throughout many cell generations. The molecular machinery that replicates the DNA – acting at the so-called replication fork – can be frequently hindered by obstacles of both extracellular and intracellular origin, such as chemicals or UV radiations on one hand, and collision with other processes occurring on the DNA, like gene transcription, on the other. These challenges to DNA replication may lead to transient slowing or stalling of replication forks: we refer to this as replication stress. Cells evolved a variety of mechanisms, which fall under the definition of DNA damage response, that allow them to respond to replication stress. Failure of these mechanisms can lead to DNA damage, and ultimately result in genomic instability, a major driving force of cancer. On the other hand, many chemotherapeutic compounds are designed to induce replication stress, exploiting the stringent requirement of highly replicating cancer cells to continuously replicate their genome. Therefore, investigating the mechanisms underlying replication stress has emerged as a key tool to both understand cancer onset, and to develop new therapeutic approaches.
Our laboratory has recently reported that, upon cellular exposure to genotoxic treatments, replication forks slow down and are frequently remodeled to form a detectable four-way junction at the replication fork, called reversed fork. This transient molecular transaction is considered to be a protective response, required to limit fork breakage in conditions of replication stress. In addition, our laboratory reported that, besides exogenous genotoxic treatments, fork reversal frequently occurs also in response to endogenous molecular processes, which are known to undermine genomic integrity, such as the activation (overexpression or amplification) of cellular proto-oncogenes.
In the current thesis, I will present our efforts to understand what triggers reversed fork formation in different contexts, and what are the factors that contribute to its formation and stability.
In the first part, I describe strategies we designed for the generation of new inducible oncogene overexpression systems in different cellular models, with the aim of describing the common or different molecular consequences downstream of aberrant activation of different oncogenes. This part has led to date to inconclusive results, mainly due to technical difficulties in establishing an efficient and robust oncogene induction system. The importance of this question (oncogene- induced replication stress in cancer) demands additional future work, and fine-tuning of the techniques for a time-controlled oncogene overexpression.
In the second part, I successfully investigated the function in replication fork remodeling of the proteins involved in homologous recombination, with particular attention on the tumor suppressors RAD51 and BRCA2. Besides their established role in double strand break repair via homologous recombination, RAD51 and BRCA2 were in fact known to protect stalled replication forks from extensive nucleolytic degradation. Underlying the importance of this alternative function for these genes, defects in fork protection lead to chromosomal instability, and contribute to the sensitivity of BRCA2-defective tumors to chemotherapeutics by yet-unknown mechanisms. Our results showed that RAD51 contributes to reverse fork formation. Moreover, we found that these structures are progressively degraded in the absence of BRCA2 (loss-of-function mutations in BRCA2 are a common feature of many cancer types). Inhibiting MRE11 nuclease activity or its recruitment to the reversed fork can restore fork integrity and prevent chromosomal breakage. On the contrary, preventing fork degradation by impairing the formation of reversed forks, leads to increased chromosomal breakage in BRCA2-defective cells, being thus detrimental for genome stability. Collectively, our study reveals that fork reversal has a crucial physiological relevance in protecting genome stability upon replication stress, and that a complex interplay of HR factors co- operate to remodel and stabilize stalled DNA replication forks.

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Additional indexing

Item Type:Dissertation
Referees:Lopes Massimo, Sartori Alessandro A, Pruschy Martin
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
Place of Publication:Zürich
Date:2017
Deposited On:07 Feb 2018 16:10
Last Modified:27 Aug 2018 09:09
Number of Pages:116
OA Status:Green

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