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
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
