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
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
The integrity of DNA is constantly threatened by large number of exogenous and endogenous agents of both chemical and physical nature. Living cells therefore posses several DNA repair mechanisms,... Show moreThe integrity of DNA is constantly threatened by large number of exogenous and endogenous agents of both chemical and physical nature. Living cells therefore posses several DNA repair mechanisms, that can detect and repair damaged DNA. One such mechanism is Nucleotide Excision Repair (NER). NER detects damaged DNA and subsequently removes a ~30nt oligonucleotide containing the damage. Repair is completed by synthesizing new DNA using the undamaged strand as a template. This thesis is focussed on the damage recognition step of NER, using the yeast Saccharomyces cerevisiae as a model organism. The Rad4 protein functions as a damage-sensor for NER. Here, experiments are described which show that two previously unidentified proteins, now named Rad33 and Rad34, are also involved in damage recognition. Rad34 is a Rad4 homologue specifically involved in NER in the ribosomal DNA (rDNA) region. Rad33 is a small protein that binds to Rad4 and to Rad34 and is essential for proper functioning of these proteins in NER. Further studies reveal that Rad33 is involved in regulating post-translational modifications of Rad4 and that Rad33 shows functional resemblance to the human Centrin2 protein, which functions in complex with the human Rad4 homologue XPC. Show less
