Mismatch repair (MMR) is a DNA repair system that corrects base-base misincorporations generated by the replicative DNA polymerases. Previous publications have shown the involvement of the MMR... Show moreMismatch repair (MMR) is a DNA repair system that corrects base-base misincorporations generated by the replicative DNA polymerases. Previous publications have shown the involvement of the MMR pathway in control of DNA damage responses. The work presented in this thesis suggests that the regulation of DNA damage induced mutagenesis and signaling may lie in the complex interplay between translesion synthesis (TLS), a subset of error-prone polymerases capable of replicating damaged DNA, and MMR. This thesis suggests that MMR may reduce DNA damage induced mutagenesis via two ways: (i) recruit the somewhat less error-prone TLS polymerases to the DNA lesion and (ii) post-replicative removal of mismatched nucleotides. Of note, inheriting a heterogenous defect in an MMR gene results in increased risk of developing cancer, in particular colorectal cancer. The reason for this specific cancer tropism is, as of yet, unclear. The colorectal tract is continuously exposed to DNA damaging agents and indeed data in this thesis shows that MMR is important in protecting against diet-derived DNA damaging agents by controlling DNA damage induced mutagenesis and signaling. As such the colorectal cancer tropism of MMR of LS may partly be explained by the role of MMR in controlling the DNA damage 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
Double-strand breaks (DSBs) are one of the most lethal forms of DNA damage. To prevent this, cells have evolved complex and highly conserved systems to detect these lesions, signal their presence,... Show moreDouble-strand breaks (DSBs) are one of the most lethal forms of DNA damage. To prevent this, cells have evolved complex and highly conserved systems to detect these lesions, signal their presence, trigger various downstream events and finally bring about repair. Two main pathways are used for DNA DSB repair: Homologous Recombination (HR) and Non-Homologous End-Joining (NHEJ). Both of them function together to maintain genome integrity. At least two NHEJ pathways have been identified: the classic NHEJ pathway (c-NHEJ) and the backup-NHEJ pathway (b-NHEJ) also called alternative-NHEJ (a-NHEJ) or microhomology-mediated end-joining (MMEJ). Agrobacterium tumefaciens is widely used as a vector to produce genetically modified plants. Agrobacterium-mediated genetic transformation involves the transfer of T-DNA from its tumor-inducing plasmid to the host cell nucleus, where it integrates into the plant genome. However, the molecular mechanism of T-DNA integration is still unclear. T-DNAs can integrate at artificially induced DSBs, which suggests that DSB repair mechanisms are probably involved in T-DNA integration in plants. Arabidopsis NHEJ mutants have subsequently been studied for T-DNA integration. However, the results obtained by different research groups were variable and revealed either no or limited negative effects. Show less
DNA is arguably the most important molecule found in any organism, as it contains all information to perform cellular functions and enables continuity of species. It is continuously exposed... Show more DNA is arguably the most important molecule found in any organism, as it contains all information to perform cellular functions and enables continuity of species. It is continuously exposed to DNA-damaging agents both from endogenous and exogenous sources. To protect DNA against these sources of DNA damage various DNA-repair mechanisms have evolved. If not properly repaired, DNA damage can lead to mutations that may eventually lead to cell-death or tumorigenesis. One of the most dangerous types of DNA damage is a DNA double-stranded break (DSB), in which a DNA molecule is broken into two pieces. Cells are equipped with several DSB-repair mechanisms to deal with this type of damage. Some of these mechanisms repair DSBs in an error-free fashion, while others are error-prone and can lead to the accumulation of mutations. Although accumulating many mutations in cells can lead to severely reduced cellular fitness, perfect DNA repair is less desirable in the long term as mutations allow for speciation and evolution to take place. The key question addressed in my thesis is which DSB-repair mechanisms organisms use to protect their genome against DSBs and I find alternative end-joining of DNA breaks to play a major role in maintaining genome stability. Show less
Our genome contains, in the form of DNA building blocks, the necessary information for orchestrating all cellular functions and ensuring species continuity. However, though being chemically stable,... Show moreOur genome contains, in the form of DNA building blocks, the necessary information for orchestrating all cellular functions and ensuring species continuity. However, though being chemically stable, our DNA is under constant assault by various endogenous or exogenous sources. To counteract the perils arising due to DNA damage and to ensure genome integrity, life has evolutionarily acquired an arsenal of protective mechanisms, amongst which prominent is DNA repair. Depending on the nature of the inflicted DNA damage, specialised DNA repair pathways are activated in order to restore the original genetic information. During this work I focused on the elucidation of DNA repair- or other cytoprotective-mechanisms ensuing upon the induction of primarily DNA Double Strand Breaks (DSBs) or secondarily helix-distorting lesions. DSBs are mainly repaired by two pathways: error-prone Non-Homologous End-Joining (NHEJ) and error-free Homologous Recombination (HR). On the other hand, helix-distorting lesions, such as those arising due to UV-irradiation or cisplatin-induced mono-adducts, are rectified by Nucleotide Excision Repair (NER). More specifically, I put emphasis on the role of protein-ubiquitylation, a widespread protein Post-Translational Modification (PTM), in the spatiotemporal regulation of an efficient DSB response. Show less
The genetic code of life is stored in DNA molecules that consist of two parallel strands of coupled nucleotides that form a DNA double helix. One of the most deleterious forms of DNA damage is a... Show moreThe genetic code of life is stored in DNA molecules that consist of two parallel strands of coupled nucleotides that form a DNA double helix. One of the most deleterious forms of DNA damage is a DNA double-strand break (DSB) in which both strands of the helix are broken. When not repaired adequately DSBs can lead to extensive loss of genetic information and/or genomic rearrangements, ultimately fueling genome instability, cellular dysfunction and malignant transformation. This thesis describes several studies conducted to examine how living organisms preserve their genetic material and how different DNA repair pathways influence genome stability. To study these questions the nematode C. elegans was used as a model organism, as it allows efficient genetic manipulation as well as in-depth genetic analysis of mutagenic processes. We exploited these unique attributes to i) convert these animals into in vivo sensors of DNA damage ii) identify factors not implicated in genome stability before, iii) unveil mechanisms that dictate DNA repair pathway choice, and iv) determine the biological consequences of endogenous barriers that impede DNA replication. Show less
In this thesis I describe the developmental role of the Y-family polymerases Pol Eta, Pol Kappa and Rev1 in protection against exogenous and endogenous damage in C. elegans. Furthermore I identify... Show moreIn this thesis I describe the developmental role of the Y-family polymerases Pol Eta, Pol Kappa and Rev1 in protection against exogenous and endogenous damage in C. elegans. Furthermore I identify a new role for the A-family Polymerase Pol Theta in repair of replication-associated breaks. Show less
This thesis, titled __Genetic and pharmacogenetic determinants of cardiovascular disease__ is divided in three sections. In section one the genetic determinants of coronary restenosis are explored.... Show moreThis thesis, titled __Genetic and pharmacogenetic determinants of cardiovascular disease__ is divided in three sections. In section one the genetic determinants of coronary restenosis are explored. In the first genome-wide association study on this condition, in the GENetic DEterminants of Restenosis study, we describe a novel locus on chromosome 12 possibly associated with restenosis. Furthermore, by using several analysis tools on this data, we describe multiple biological pathways and genes that are likely associated with restenosis. In section two, we focus on genetic factors involved in three other (cardio)vascular diseases. We explore the role of DNA repair genes in myocardial infarction and stroke and the genetic determinants of dialysis shunt failure. Section three is on pharmacogenetics. In particular, we were interested in genetic variation involved in aspirin and clopidogrel resistance. We validated two genetic polymorphisms associated with recurrent thrombotic events during treatment with these agents in patients with an acute myocardial infarction. Genetic research is a fast developing field of research. By increasing our knowledge on the molecular background of diseases, genetics potentially could lead to more personalized treatment in the near future. 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
Nucleotide Excision Repair (NER) is a conserved DNA repair pathway capable of removing a broad spectrum of DNA damage. In human cells a defect in NER leads to the disorder Xeroderma pigmentosum (XP... Show moreNucleotide Excision Repair (NER) is a conserved DNA repair pathway capable of removing a broad spectrum of DNA damage. In human cells a defect in NER leads to the disorder Xeroderma pigmentosum (XP). The yeast Saccharomyces cerevisiae is an excellent model organism to study the mechanism of NER. The yeast proteins Rad4 and Rad23 are important in NER and involved both Transcription-Coupled and Global Genome NER sub-pathways. Chapter 2 describes a novel mechanism of generegulation by the GG-NER E3 ligase. This protein complex can regulate dNTP synthesis via UV-induced Rad4 ubiquitination. In Chapter 3 we describe the effect of this histone on TC-NER at the rDNA. We find that Rad34 is only required when Histone H1 is present. In Chapter 4 we analyze the phenotype of a yeast strain containing a RAD4 mutation. The mutation affects a conserved residue that when altered in the human homolog XPC, leads to the cancer prone disorder XP. Chapter 5 is dedicated to the Rad4-Rad23 interaction that we describe in more detail. We uncovered a novel N-terminal interaction of Rad23 with Rad4 that is important for TC-NER. Finally, in Chapter 6 we reveal an unexpected UV phenotype for the commonly used wild-type yeast strain WCG4A. In this strain we identified the mutation to reside in the RAD4 gene Show less
Transplant recipients generally require lifelong treatment with immunosuppressive medication to prevent rejection of the graft by their immune system. Inhibitors of the enzyme calcineurin,... Show moreTransplant recipients generally require lifelong treatment with immunosuppressive medication to prevent rejection of the graft by their immune system. Inhibitors of the enzyme calcineurin, including cyclosporin A and tacrolimus, constitute a very potent class of immunosuppressants that has revolutionized transplant medicine. However, their reputation has been showing cracks due to the severe side-effects associated with long-term use of these drugs, including an explosively increased risk of developing skin cancer. The pathophysiological mechanism of this phenomenon is not known, although a number of hypotheses have been put forward. In this dissertation, we show that oxidative stress, mainly derived from exposure to UVA radiation, may locally augment the effects of the calcineurin inhibitors; we propose that overly strong suppression of calcineurin activity may result in malignancy formation due to disruption of tumor-suppressive signaling pathways or disturbed immunosurveillance in skin. 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
Repair of damage in the DNA is essential for an organism. Therefore, several repair mechanisms have evolved. In this thesis, the mechanism of Transcription-Coupled Nucleotide Excision Repair (TC... Show moreRepair of damage in the DNA is essential for an organism. Therefore, several repair mechanisms have evolved. In this thesis, the mechanism of Transcription-Coupled Nucleotide Excision Repair (TC-NER) and the UV Damage Endonuclease repair pathway (UVDE) have been studied. Central to TC-NER is the protein Cockayne Syndrome protein A (CSA). Its biological importance can be seen in that mutations in CSA cause the human, serious disorder Cockayne Syndrome. This thesis describes structural and biochemical studies of this protein, which give insights into its substrate-binding and into how mutations in this protein cause the disease Cockayne Syndrome. Biochemical and structural studies of UVDE show the identity and role of its post-translational modification, a carboxylation. A cocrystal structure of UVDE with 6-4PP DNA shows how UVDE can recognize UV damaged DNA. Show less
Cancer is caused by an accumulation of mutations (formed when cells attempt to replicate damaged DNA) that lead to unchecked cell growth and proliferation. The first chapter of this thesis gives an... Show moreCancer is caused by an accumulation of mutations (formed when cells attempt to replicate damaged DNA) that lead to unchecked cell growth and proliferation. The first chapter of this thesis gives an overview of the major DNA-damaging agents and the opposing repair pathways, subsequently nucleotide excision repair (NER), able to repair a wide range of damages, is discussed. The second chapter thoroughly discusses the assembly of the NER complex, subsequent incision and repair synthesis. The recently elucidated regulation of NER is also discussed in detail whereas DNA-damage induced signalling is discussed briefly. Perspectives in respect to NER related research are discussed in the third chapter. Chapter four concerns the regulation of NER, preventing dual incision when gaps, formed by previously excised damages, cannot be filled. Chapter five and six concern the recruitment and activity of replication factors after dual incision. Finally, chapter seven describes UV-damage mediated incision and signalling in NER deficient cells. The main focus of this thesis is the sequence of events following dual incision, as much was known about the steps leading to incision yet little was known about the handover from pre- to post-incision complexes, the recruitment of post-incision factors and how they function in NER Show less
DNA is continuously exposed to exogenous and genotoxic insults including ionizing and ultraviolet radiation as well as chemical agents. DNA damage can compromise the integrity of the genome and... Show moreDNA is continuously exposed to exogenous and genotoxic insults including ionizing and ultraviolet radiation as well as chemical agents. DNA damage can compromise the integrity of the genome and have potentially deleterious effects. Ultraviolet light (UV) can induce the formation of helix distorting lesions such as 6-4 photoproducts (6-4PP) and cyclopyrimidine dimers (CPD). In humans, the nucleotide excision repair (NER) pathway is solely responsible for the repair of these lesions. A defect in NER can lead to extreme sun sensitivity and an elevated risk of developing skin cancer as observed in patients with the inherited disorder xeroderma pigmentosum. The work described in this thesis focuses on NER in human cells. Findings include: 1) UV-DDB stimulates the repair of photolesions, 2) UV-DDB binds to UV lesions independently of XPC suggesting it may have a role in chromatin priming, 3) the sealing of DNA nicks during NER are entirely on XRCC1-DNA ligase III_ complex in quiescent cells, 4) RPA couples NER mediated incision to DNA repair synthesis and ligation, 5) Arsenic induces its co-carcinogenic effects at least in part by disrupting the sealing of DNA nicks that arise during NER. Show less
Evidence is accumulating that infliction of DNA damage can lead to spatial repositioning of chromatin. Mitomycin C (MMC)-induced interchanges between homologous chromosomes have been taken as... Show moreEvidence is accumulating that infliction of DNA damage can lead to spatial repositioning of chromatin. Mitomycin C (MMC)-induced interchanges between homologous chromosomes have been taken as cytological evidence for somatic pairing of human chromosomes. Pairing of specific chromosomal regions might differ dependent on biological states or activities, i.e. cell type, cell cycle phase, differentiation stage and diseases. However, the biological relevance of pairing is unclear. The aim of this thesis was to dissect the mechanisms that underlie the interaction between homologous chromosomes observed in metaphase and the interphase of the cell cycle. To reach this goal, we assessed the effect of genotoxic agents such as X-rays, UV radiation and MMC on the positioning of homologous chromosomal regions within the nucleus of human cells. Moreover, we examined the relationship between interphase pairing and processing of DNA damage that leads to exchange formation. We found that: (i) pairing of heterochromatic regions is a general response to genotoxic stress, (ii) pairing is correlated with exchanges between homologous chromosomes after exposure to the anticancer drug mitomycin C (MMC), (iii) pairing is genetically controlled by genes involved in the DNA damage response and (iv) processing of MMC-induced DNA damage is initiated in non-dividing cells. Show less
The initial damage recognizing complex in bacterial nucleotide excision repair consists of two UvrA and two UvrB molecules. Of these proteins UvrB is the main damage recognition protein and is... Show moreThe initial damage recognizing complex in bacterial nucleotide excision repair consists of two UvrA and two UvrB molecules. Of these proteins UvrB is the main damage recognition protein and is capable of recognizing various structurally unrelated types of damage. To do so, the protein must recognize a common alteration of the DNA structure induced by these different damages. For UvrB sterical hindrance of the present DNA modification prevents it from passing behind a _-hairpin structure present in the protein, thereby conferring damage recognition. Upon stalling of the protein at the site of damage in this way we show that two nucleotides become extrahelical: the nucleotide directly 3__ to the lesion and its base-pairing partner in the non-damaged strand. In contrast to other repair enzymes however the damaged nucleotide itself does not become extrahelical. Flipping in one of the two DNA strands has been shown to be important in preventing stable binding of the protein to undamaged DNA. Flipping in the other DNA strand however is required for efficient 3__ incision by UvrC, the nuclease of the system. A second incision at the 5__ side of the damage by the same protein facilitates removal of the damage-containing DNA oligonucleotide. Show less
The results presented in this thesis provide new information on the role of the p53.S389A point mutation in chemical-induced tumorigenesis. After DNA damage, p53 protein levels increase due to... Show moreThe results presented in this thesis provide new information on the role of the p53.S389A point mutation in chemical-induced tumorigenesis. After DNA damage, p53 protein levels increase due to several post-translational activation processes. Phosphorylation of p53.S389 seems to be partly required for optimal induction of these p53 protein levels. Next, target genes are either induced or repressed, and phosphorylation of p53.S389 seems essential for an optimal p53-related transcriptional response both endogenously (especially repressed genes) as well as after the induction of DNA damage. Than as a read-out system for the activation of different genes, several cellular responses (apoptosis, cell cycle arrest etc.) can be observed, which again seems partly dependent on p53.S389 phosphorylation. When these processes are adversely affected due to inadequate functioning of p53, like is the case in p53.S389A mutant mice, this might lead to increased risks of developing tumors. Indeed, two chronic carcinogenicity experiments revealed an increased sensitivity of the p53.S389A mutant mice for tumor development upon exposure to DNA damaging agents. In conclusion, knowledge about the in vivo relationship between DNA damage induction, regulation of p53 activity (in terms of cell cycle control and/or apoptosis), DNA repair (NER) and the development of cancer was obtained. Show less
Of all exogenous agents that damage genomic DNA and hence threaten its integrity, the ultraviolet B (UVB) component of sunlight is highly relevant because of its abundance. UVB induces... Show moreOf all exogenous agents that damage genomic DNA and hence threaten its integrity, the ultraviolet B (UVB) component of sunlight is highly relevant because of its abundance. UVB induces predominantly cyclobutane pyrimidine dimers and 6-4 photoproducts. In humans, these photolesions are repaired by the nucleotide excision repair (NER) system. Additionally NER removes a range of bulky, helix-distorting lesions, including chemical adducts caused by the anticancer drug cisplatin and complex hydrocarbons found in burnt food and cigarette smoke. Patients suffering from xeroderma pigmentosum, an inherited disorder caused by mutations in genes encoding NER proteins, display hypersensitivity to sunlight and a dramatically increased incidence of skin cancers. The research in this thesis concerns NER in intact human cells. Findings include: 1) the NER complex assembles on a lesion in a sequential manner, as opposed to pre-existing as a holo-complex; 2) TFIIH is not involved in the UV-induced inhibition of transcription; 3) XPA is not complexed with RPA, contradicting previous reports; 4) UV-DDB stimulates the repair of low levels of 6-4 photoproducts; and 5) pol_, pol_ and ligase I are involved in the DNA resynthesis step of NER in vivo. Show less
