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
