Cells constitute the tissues of our body and are responsible for producing various changes in response to different situations. For instance, the repair of damaged DNA. DNA resides within the cell... Show moreCells constitute the tissues of our body and are responsible for producing various changes in response to different situations. For instance, the repair of damaged DNA. DNA resides within the cell nucleus and can be transcribed and translated into proteins, which play vital roles in numerous cellular processes. The cell relies on modifying existing proteins to carry out essential functions. These modifications can involve the conjugation of small molecules such as Ubiquitin (Ub) or Small Ubiquitin-like Modifiers (SUMOs), leading to protein degradation, conformational changes or intracellular relocation of critical proteins. The conjugation of these small molecules involves a well-orchestrated sequence of enzymatic activities performed by dedicated enzymes: E1 (activating), E2 (conjugating) and E3 (ligase). Among these, the E3 ligase enzymes hold significant importance as they confer substrate specificity.In this thesis, we have developed an advanced Mass-Spectrometry technology called TULIP2 (Targets for Ubiquitin Ligases Identified by Proteomics 2), which facilitates the identification of Ubiquitination targets for specific E3 ligases of interest. Using this technology, we have investigated the BRCA1-BARD1 E3 ligase and explore the in vivo role of the E2 UBE2D3. Furthermore, we have adapted the TULIP2 technology to create the SUMO Activated Target Traps (SATTs), enabling the identification of an E3-specific SUMO proteome. Show less
All cellular organisms contain genomic DNA which provides the instructions for their correct development and functioning. Damage to this DNA may interfere with critical cellular processes such as... Show moreAll cellular organisms contain genomic DNA which provides the instructions for their correct development and functioning. Damage to this DNA may interfere with critical cellular processes such as transcription and replication and has the potential to drive mutagenesis. In turn, this may underlie inherited disorders and accelerate progression of diseases such as cancer and neurodegenerative disorders. The protection of cells and organisms against these devastating effects of DNA damage relies on the DNA damage response (DDR), which comprises a complex network of signaling and repair pathways that coordinate the sensing, signaling and repair of DNA lesions while accommodating suitable adjustments in for instance chromatin structure and cell cycle progression. Not only does the DDR dictate the appropriate repair pathway for several types of DNA damage, including DNA double-strand breaks (DSB), it also modulates replication fork surveillance mechanisms in response to DNA replication stress (RS). While many core proteins have been studied in detail, the full repertoire of factors involved in these pathways remains unknown. Clearly, extending our knowledge on regulators of the DDR will contribute to our understanding of the development, and possibly the treatment, of the numerous disorders that are associated with defects in the DDR. The research described in this thesis has successfully identified and characterized novel factors in DSB repair and the RS response. Show less
This thesis aims to gain a better understanding of NER, to elucidate new molecular mechanisms and proteins that orchestrate how DNA repair is carried out on genomic DNA that is tightly packed in... Show moreThis thesis aims to gain a better understanding of NER, to elucidate new molecular mechanisms and proteins that orchestrate how DNA repair is carried out on genomic DNA that is tightly packed in chromatin inside the living cell. It is important to obtain a better clinical picture of how inherited defects in DNA repair genes shapes phenotypes in patients with DNA repair-deficiency disorders. Show less
The cellular response to transcription-blocking DNA lesions involves the stalling of elongating RNA Polymerase II (RNAPIIo) at the lesion as well as a global shutdown of transcription. The stalling... Show moreThe cellular response to transcription-blocking DNA lesions involves the stalling of elongating RNA Polymerase II (RNAPIIo) at the lesion as well as a global shutdown of transcription. The stalling of RNAPIIo at such lesions initiates the transcription-coupled nucleotide excision repair pathway (TCR) to efficiently remove the damage and restore transcription. The TCR proteins, CSB, CSA, and UVSSA, are essential for the repair of transcription-blocking DNA lesions, but how the interplay between these proteins targets the core repair machinery, including the TFIIH complex, to lesion stalled RNAPIIo remains largely unknown.Here, we demonstrate a sequential and highly cooperative assembly of TCR proteins and unveil the mechanism for TFIIH recruitment to DNA damage-stalled RNAPIIo. Importantly, we identified the previously uncharacterized ELOF1 gene as a core TCR factor with an additional role in preventing DNA damage during DNA replication. Show less
DNA encodes the genetic instructions for living organisms. However, damage to the DNA is inevitable, because DNA itself is an unstable molecule and environmental factors such as UV-radiation or X... Show moreDNA encodes the genetic instructions for living organisms. However, damage to the DNA is inevitable, because DNA itself is an unstable molecule and environmental factors such as UV-radiation or X-rays cause damage to the DNA. A certain type of DNA damages can block DNA replication, an essential step before cell can divide. The polymerases that normally replicate DNA are incredibly efficient and virtually flawless on undamaged DNA, but they cannot replicate damaged DNA. In multi-celled organisms, the most important defense mechanism against this is Translesion DNA synthesis (TLS). TLS protects against various negative consequences of damage to the DNA. For this, TLS utilizes specialized TLS polymerases that can replicate damaged DNA.My experiments show that the strong evolutionary conservation of TLS is explained by the dual functions of TLS: guarding replication potential and genome stability. TLS suppresses genomic instability, by preventing conversion of replication blocks to double-stranded DNA breaks (DSBs). Without functional TLS, DSBs arise and result in larger and more harmful mutations. TLS is beneficial for organisms because it supports continuous reproduction and growth. Although DNA damage is always present and unavoidable, TLS guards against the formation of mutations that would otherwise lead to cancer, aging and congenital disease. Show less
Inactivating mutations in BRCA1 or BRCA2 genes predispose to several types of cancer. Owing to their roles in maintaining genomic stability, lack of BRCA1/2 results in DNA damage repair defects, a... Show moreInactivating mutations in BRCA1 or BRCA2 genes predispose to several types of cancer. Owing to their roles in maintaining genomic stability, lack of BRCA1/2 results in DNA damage repair defects, a vulnerability that can be exploited therapeutically by the inhibition of poly(ADPribose) polymerase 1 (PARP1). Unfortunately, clinical benefit of PARPi therapy is often limited by emerging drug resistance. Identification of PARPi resistance mechanisms is therefore crucial to improve the clinical outcome and design strategies that would ultimately prevent or target resistant tumors.The use of genetically engineered mouse models (GEMMs) of BRCA1/2-associated breastcancer in this work has allowed us to model PARPi resistance in vivo in well-defined genetic contexts. By combining high-throughput genetic screens, multiple omics analyses and functional assays, we identified several factors of PARPi resistance and explained their role in therapy failure. Moreover, we established a new tumor-derived organoid system thatenables robust in vivo validation of putative drug resistance factors. Finally, work described in this thesis has advanced our understanding of basic biological processes involved in DNA damage signaling and repair. Show less
In this thesis, I aimed at decoding the role of SUMO in the DNA damage repair pathway. The SUMO system is believed to be involved in this process at several levels. I focused on the most inevitable... Show moreIn this thesis, I aimed at decoding the role of SUMO in the DNA damage repair pathway. The SUMO system is believed to be involved in this process at several levels. I focused on the most inevitable DNA obstacle causing DNA replication stress, and the cellular roles of SUMOylation in repairing DNA replication stress caused DNA damage. Post-translational modifications are essential regulators of proteins. PTMs do not only play their roles solo but extensively interact with each other. Our knowledge about proteins modified by a combination of SUMO and ubiquitin, SUMO and phosphate and crosstalk between them is quite limited. This thesis also aimed at deciphering the crosstalk between SUMOylation and phosphorylation and ubiquitination during the DNA damage response and searching for indirect and direct targets for the human STUbL RNF4, which mediates the ubiquitination of SUMOylated target proteins. Lastly, we adopted the strategy described for SUMO and introduced His10-tagged UFM-1-K0 to identify UFM-1 acceptor lysines. We identified and confirmed RPL26 as a key UFM1 target and further confirmed that the UFMylated form of RPL26 can efficiently interact with the Signal Recognition Particle Receptor, implicating that UFMylation could regulate protein transfer to the Endoplasmic Reticulum. Show less
To protect the genome, cells have evolved a diverse set of pathways designed to sense, signal, and repair multiple types of DNA damage. To assess the degree of coordination and crosstalk among... Show moreTo protect the genome, cells have evolved a diverse set of pathways designed to sense, signal, and repair multiple types of DNA damage. To assess the degree of coordination and crosstalk among these pathways, we systematically mapped changes in the cell's genetic network across a panel of different DNA-damaging agents, resulting in ~1,800,000 differential measurements. Each agent was associated with a distinct interaction pattern, which, unlike single-mutant phenotypes or gene expression data, has high statistical power to pinpoint the specific repair mechanisms at work. The agent-specific networks revealed roles for the histone acetyltranferase Rtt109 in the mutagenic bypass of DNA lesions and the neddylation machinery in cell-cycle regulation and genome stability, while the network induced by multiple agents implicates Irc21, an uncharacterized protein, in checkpoint control and DNA repair. Our multiconditional genetic interaction map provides a unique resource that identifies agent-specific and general DNA damage response pathways. Show less
Genetic alterations such as mutations, genomic rearrangements and aneuploidy, are commonly observed in tumors. To counteract this cells have multiple genome maintenance and surveillance systems to... Show moreGenetic alterations such as mutations, genomic rearrangements and aneuploidy, are commonly observed in tumors. To counteract this cells have multiple genome maintenance and surveillance systems to minimize the rate at which genomic alterations arise. The aim of the thesis is to gain understanding of processes and pathways that contribute to the maintenance of genome stability and to establish how defects in these processes and pathways abrogate the DNA damage response and consequently may promote genomic instability and development of cancer. The work described in this thesis addresses various aspects of the cellular response of mammalian cells to DNA damaging agents including changes in post translational modifications that occur after genotoxic stress. The role of poly-adenosyl ribose modification in nucleotide excision repair is investigated and found to be important for the recruitment of a chromatin remodeling protein and repair. Phosphoproteomic analysis identified changes in the global phosphorylation state of proteins following genotoxic stress. Key kinases responding to DNA damage are ATR and related kinases. A detailed study into the requirements for ATR activation after UV exposure indicates that at least two distinct modes of activation exist. Finally we describe the profound sensitivity of Cornelia de Lange Syndrome cells to DNA damage. Show less
DNA damage, mutations and genomic instability are established driving forces of cancer and other age-related diseases. Mutations in tumor suppressor genes and oncogenes are very frequently found in... Show moreDNA damage, mutations and genomic instability are established driving forces of cancer and other age-related diseases. Mutations in tumor suppressor genes and oncogenes are very frequently found in tumors and genomic instability is the most common enabling characteristic of cancer. Aging is also believed to be enabled, amongst others, by genomic instability. DNA repair pathways, like the nucleotide excision repair (NER) pathway and cell cycle control (e.g. p53-dependent) processes are therefore vital to organisms, since these processes counteract or prevent genomic instability, and are thought to underlie, when affected, aging and age-related diseases like cancer. To unravel the functions, mechanisms and pathways involved in the onset of aging and age-related diseases we have investigated several mouse models deficient in either DNA repair (NER) capacity (Chapter 3, 4), cell cycle control (p53) (Chapter 6) or both (Chapter 5), and compared this to a wild type situation (Chapter 2). The use of mouse models enabled us to investigate cancer and aging in a controlled environment, minimizing possible confounding factors. Additionally, the mouse models can be useful as an alternative tool to identify genotoxic and non-genotoxic carcinogens that can be harmful to the society and the environment (Chapter 5). Show less
During my PhD project, I studied the role of several chromatin remodelers in the DNA double strand break (DSB) response. We discovered that both CHD4 and SMARCA5 are required for ubiquitin... Show moreDuring my PhD project, I studied the role of several chromatin remodelers in the DNA double strand break (DSB) response. We discovered that both CHD4 and SMARCA5 are required for ubiquitin signaling through the E3 ubiquitin ligases RNF8 and RNF168, which is a central signaling event in the response to DSBs. Furthermore, we found that SMARCA5 actually interacts with RNF168. Both CHD4 and SMARCA5 act at the break site itself and modulate DSB repair. Additionally we found that the DSB repair protein Rad51 is essential for mouse development since Rad51C knock out mice were embryonic lethal. Show less
Cells in the human body have to deal with DNA damage daily, either caused by external or internal sources. The DDR is particularly strong in stem cells. Since these cells have a long life span and... Show moreCells in the human body have to deal with DNA damage daily, either caused by external or internal sources. The DDR is particularly strong in stem cells. Since these cells have a long life span and are essential for tissue homeostasis, tolerance to damaged DNA would lead to accumulation of mutations and malignant transformation. In addition, accumulation of damaged DNA would lead to loss of the stem cell pool and contribute to aging. In this thesis I investigated the role of the DNA damage response in the context of stem cells as well as cancer cells, from the response to different DNA damaging agents, to the importance of the interaction with the extracellular matrix in combination with the presence of oncogenes. In order to acquire a complete picture of the DNA damage response in mES cells, and therefore elucidate novel pathways involved in this particular response, we combined OMICS techniques such as Functional Genomics, Transcriptomics and Phosphoprotoemics, that once overlapped, allowed us to find novel pathways that where not previously described to be involved in the DNA damage response. Show less
In this thesis we attempt to provide a better understanding of the principles that underlie the spatial dynamic organization of the cell nucleus. Chapter 1 reviews the current status of knowledge... Show moreIn this thesis we attempt to provide a better understanding of the principles that underlie the spatial dynamic organization of the cell nucleus. Chapter 1 reviews the current status of knowledge about the structural and functional organization of the cell nucleus. In chapter 2, the development of a computer program is described that has been designed to track the 2D and 3D motion of objects in the nucleus of living cells. In chapter 3, evi-dence is provided for the existence of a nuclear matrix structure that is composed of lamin proteins, emerin and actin. By analyzing the dynamics of telomeres in nuclei of cells showing reduced levels of lamin expression, it is investigated whether telomeres anchor to an inner nuclear lamina structure. In chapter 4 the de novo formation of PML nuclear bodies is described. Using live cell imaging and immunocytochemistry it is dem-onstrated that telomeres play a role in the de novo formation of PML bodies. In chapter 5 it is investigated whether nuclear bodies are associated with chromatin in the cell nucleus. After treating cells with DNA alkylating agent MMS, the dynamics of PML bodies, Cajal bodies and speckles has been analyzed relative to chromatin in the 3D space of the cell nucleus Show less
The work presented in this thesis has focused on the role of Mitogen Activated Protein Kinases (MAPKs) and their major downstream targets, the AP-1 transcription factors, in particular the AP-1... Show moreThe work presented in this thesis has focused on the role of Mitogen Activated Protein Kinases (MAPKs) and their major downstream targets, the AP-1 transcription factors, in particular the AP-1 components ATF3, Fra1, c-Jun, ATF-2 and c-Fos. Chapter II provides information on the signaling pathways involved in the activation of ATF-2 and ATF3 in the response of primary human fibroblasts to ionizing radiation. In chapter III c-Jun and ATF3, the MAPK JNK and the MAPK-phosphatase MKP-1 are identified as important sensors of UV-induced-DNA damage in transcribed genes. Chapter IV shows that ATF3 acts as an antiapoptotic JNK target in T98G glioblastoma cells, whereas Fra1 seems to act as a proapoptotic effector of both JNK and ERK. In addition, it is shown that ATF3 and Fra1 have opposite effects on cisplatin-induced S phase arrest. Chapter V shows that Fra1 also can exhibit a pro-apoptotic function in UV-irradiated fibroblasts. Furthermore, this chapter reports an as yet unknown function of JNK: repression of the transactivating activity of c-Jun/Fos(- like) dimers, mediated via hyper-phosphorylation of the c-Jun transactivation domain. The data further emphasizes that c-Jun/Fos(-like) and c-Jun/ATF dimers and their respective target genes can exhibit opposite functions in DNA damage responses. Show less
Sunlight has many beneficial effects. However, from a biological point of view, solar UV radiation has also detrimental effects, especially at high doses of exposure. Because of its genotoxic... Show moreSunlight has many beneficial effects. However, from a biological point of view, solar UV radiation has also detrimental effects, especially at high doses of exposure. Because of its genotoxic properties, UV radiation plays an important role in the induction of skin cancer. In the last decennia, the incidence of skin cancer is rapidly increasing. This can partly be explained by an ageing population. However, the rise in skin cancer incidence appears to be primarily due to a change in human lifestyle in which sunbathing has become very popular. Our skin is continuously challenged by UV radiation, which may lead to irreversible damage. In order to withstand sustained physical, chemical and biological damage from the environment, among which UV radiation, the skin is continuously renewed. The regenerative capacity of skin is conferred by stem cells, which persist throughout the organism__s lifetime. Because of their long residency and unlimited capacity to replicate, stem cells might accumulate DNA damage and generate the multiple genetic lesions necessary for tumour development, despite efficient cellular defence mechanisms against DNA damage. Thus, stem cells may play an important role in carcinogenesis. The aim of this study is to investigate the role of stem cells in skin carcinogenesis. Show less
