Genetics - Theses
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ItemG2 dna damage and checkpoint control(University of Melbourne, 2004)Responding to DNA damage is a fundamental function of a cells existence. Failure to do so can result in mutations that may result in a loss of viability and contribute to tumourigenesis in multicellular organisms. For this reason, cells have evolved complex signalling pathways involved in the detection and response to DNA damage. One subset of these pathways are the checkpoint signalling cascades, which act to halt cell cycle progression following DNA damage, allowing time for repair to be completed. Mutations in components of the checkpoint pathways have been shown to result in increased sensitivity to both natural and synthetic DNA damaging agents in all eukaryotes and in cancer susceptibility in humans. Several checkpoint signalling pathways exist within the cell which respond to different stimuli at various phases on the cell cycle. Of interest in this thesis is the G2 DNA damage checkpoint, which prevents mitotic entry in the presence of DNA damage. The study of checkpoint signalling and cell cycle regulation more generally, has been largely facilitated by use of the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae as model organisms. As the fission yeast S. pombe is easily grown and manipulated in the laboratory and have a prolonged G2 phase, they have been particularly useful as for the study of the regulation of mitotic entry. Despite the evolutionary distance, the genes and interactions identified in S. pombe are highly conserved in mammalian cells. To date, all the components of the G2 DNA damage checkpoint identified in S. pombe have been found to have human homologues with conserved functions. The study of both yeasts has led to the development of a sophisticated model of the response to DNA damage in the G2 phase of the cell cycle. This response can be divided into four distinct phases: the detection of DNA damage, the initiation of the checkpoint, checkpoint maintenance whilst repair is underway (which would presumably require communication between repair and checkpoint proteins) and the release of the checkpoint allowing the resumption of cell division. Many proteins have been identified that are involved in the detection of DNA damage and/or the initiation of the DNA damage checkpoint. This includes the members of the PI3K-like kinases (PIKK), ATM and ATR. PIKK family members have also been shown to communicate with many downstream targets involved in cellular processes. In S. pombe, the ATR homologue Rad3 has been shown to phosphorylate the checkpoint kinase Chk1 following DNA damage. This is a demonstrated mechanism of checkpoint initiation. Following its phosphorylation, Chk1 itself phosphorylates the phosphatase Cdc25 and the kinase Wee1. These proteins are core cell cycle regulators, whose opposing activities control mitotic entry by regulating phosphorylation of the CDK, Cdc2. Cdc2 is active when dephosphorylated and bound to its cyclin partner cyclinB, and in this state promotes mitotic entry. Chk1 activity induces the temporary stabilisation of Wee1 and inhibition of Cdc25, thereby promoting Y15 phosphorylation and a cell cycle arrest. Therefore, Chk1 represents the interface between signalling from proteins involved in the checkpoint response (Rad3) and proteins involved in the regulation of mitotic entry (Cdc25 and Wee1). Using a conditional allele of Rad3 in S. pombe, it has been shown that Rad3 function is required for the initiation but not the maintenance of the G2 DNA damage checkpoint: inactivation of Rad3 following DNA damage has no effect on cell viability or checkpoint function. This shows that checkpoint initiation and maintenance are distinct pathways. In all systems examined, from yeast to humans, the absence of Chk1 activity cells leads to DNA damage sensitivity caused by a loss of checkpoint function. So it has become clear that Chk1 is required for checkpoint initiation, but, at the commencement of this study it remained to be seen, what role if any Chk1 played in checkpoint maintenance. In this thesis I present data that shows Chk1 is required for both checkpoint initiation and maintenance. I have found that Chk1 activity is required for the duration of the G2 DNA damage checkpoint and its inactivation at any point following DNA damage leads to checkpoint failure and a consequent decrease in cell viability. It follows, that Chk1 inactivation will be an important step in resumption of the cell cycle or checkpoint release. This finding contributes to a body of data that suggests Chk1 regulation is far more complex than currently understood. Chk1 overexpression has been shown to induce a checkpoint arrest, independent of its phosphorylation and of many upstream checkpoint components. However, this requires substantial Chk1 overexpression, indicating that Chk1 is normally under strict negative regulation in the cell. Furthermore, mutations within the Chk1 C-terminal domain have been shown to have both stimulating and suppressing effects. Indicating that this area contains both positive and negative regulatory elements. Finally, Chk1 has been shown to interact with several proteins, the relevance of some of these interactions is not yet understood. Therefore, at the outset of this study it was clear that further examination of Chk1 regulation was required. In this thesis I outline the examination of a novel C-terminal mutant of Chk1 that appears to be more physiologically active than wildtype Chk1. I show that this phenotype is not produced by altered expression or increased kinase activity. Therefore I postulated that this allele (chk1-E472D) has altered interaction with regulatory proteins. In order to identify these proteins, I examine the overexpression of Chk1-E472D in the absence of several key checkpoint proteins that have been previously shown to be required for Chk1 function. Most significantly, I found that this Chk1 mutant has an altered phenotype to the wildtype protein when overexpressed in the absence of cdc25 and the 14-3-3 protein, Rad24. Finally, as the G2 DNA damage checkpoints have been found to contribute to resistance to chemotherapeutic agents in tumour cells, Chk1 inactivation has become a major target in the development of new anti-cancer regimens. Therefore, a thorough understanding of Chk1 function is especially important in order to validate this methodology and ensure the avoidance of unforeseen side effects. The contribution of this thesis to the understanding of the timing of Chk1 activity and the proteins involved in its regulation will be of assistance in this area.
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ItemNo Preview AvailableRegulation of the G2(University of Melbourne, 2000)In order to survive DNA damage, cells respond with a range of mechanisms, including DNA repair, checkpoint responses, and survival mechanisms. The DNA damage checkpoint cannot be viewed as a simple signal transduction pathway. It is divided into several steps that are genetically separable. These observations have lead to the questions that are addressed in this project. Firstly, how does the checkpoint, interact with the core G2/M cell cycle machinery. I address this with a biochemical study of the affects of a DNA damage checkpoint on Y15 regulation of p34cdc2/cyclinB. This study allows the two pathways to be linked. Secondly, do we know all the proteins that are involved in the checkpoint. We do not know how the checkpoint detects DNA damage, nor how the checkpoint integrates with signals from DNA repair pathways, and so other, as yet unidentified, checkpoint genes may fill in these gaps. In this study I used a genetic screen to identify other members of the pathway. The mutants isolated in this screen allowed the posing of new questions about the DNA damage checkpoint pathway. It is becoming increasingly clear, through this and other studies, that essential genes will play roles in the checkpoint pathway. This allows us to link the checkpoint pathway with essential pathways such as DNA replication or chromatin organisation. In a screen for new genes involved in the DNA damage checkpoint, I isolated two essential genes that play roles in G2 checkpoint responses. cut5 provides a link between DNA replication and the G2 checkpoints responding to DNA damage and blocks to DNA replication. The isolation of a novel allele of rad18 in this project is the first evidence in S. pombe of a gene linking the DNA damage checkpoint and DNA repair. Several pieces of evidence point to rad18 playing an essential role in chromatin organisation and this then links chromatin regulation to the regulation of both repair and checkpoint responses to DNA. An allele-specific suppressor of this rad18 allele, brc1, suggested a link between checkpoint and repair pathways and independent regulators of genomic stability. The novel rad18 allele also allows us to address the issue of checkpoint maintenance and checkpoint initiation. The final gene isolated in the screen was a DNA repair gene that is not absolutely required for the ability to repair. As it is not clear what repair pathway this gene is involved in, it suggests a broad-range defect, which may also link the processes of chromatin organisation and DNA repair. The isolation of these genes links together multiple pathways of DNA damage responses. It suggests that genes with essential roles in processes such as DNA replication, chromatin organisation and maintenance of genomic stability may also play roles in checkpoint and other responses. Further screening, taking advantage of the types of phenotypes discovered in this screen, will be needed to identify these genes.
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ItemNo Preview AvailableRegulation of the G2(University of Melbourne, 2000)In order to survive DNA damage, cells respond with a range of mechanisms, including DNA repair, checkpoint responses, and survival mechanisms. The DNA damage checkpoint cannot be viewed as a simple signal transduction pathway. It is divided into several steps that are genetically separable. These observations have lead to the questions that are addressed in this project. Firstly, how does the checkpoint, interact with the core G2/M cell cycle machinery. I address this with a biochemical study of the affects of a DNA damage checkpoint on Y15 regulation of p34cdc2/cyclinB. This study allows the two pathways to be linked. Secondly, do we know all the proteins that are involved in the checkpoint. We do not know how the checkpoint detects DNA damage, nor how the checkpoint integrates with signals from DNA repair pathways, and so other, as yet unidentified, checkpoint genes may fill in these gaps. In this study I used a genetic screen to identify other members of the pathway. The mutants isolated in this screen allowed the posing of new questions about the DNA damage checkpoint pathway. It is becoming increasingly clear, through this and other studies, that essential genes will play roles in the checkpoint pathway. This allows us to link the checkpoint pathway with essential pathways such as DNA replication or chromatin organisation. In a screen for new genes involved in the DNA damage checkpoint, I isolated two essential genes that play roles in G2 checkpoint responses. cut5 provides a link between DNA replication and the G2 checkpoints responding to DNA damage and blocks to DNA replication. The isolation of a novel allele of rad18 in this project is the first evidence in S. pombe of a gene linking the DNA damage checkpoint and DNA repair. Several pieces of evidence point to rad18 playing an essential role in chromatin organisation and this then links chromatin regulation to the regulation of both repair and checkpoint responses to DNA. An allele-specific suppressor of this rad18 allele, brc1, suggested a link between checkpoint and repair pathways and independent regulators of genomic stability. The novel rad18 allele also allows us to address the issue of checkpoint maintenance and checkpoint initiation. The final gene isolated in the screen was a DNA repair gene that is not absolutely required for the ability to repair. As it is not clear what repair pathway this gene is involved in, it suggests a broad-range defect, which may also link the processes of chromatin organisation and DNA repair. The isolation of these genes links together multiple pathways of DNA damage responses. It suggests that genes with essential roles in processes such as DNA replication, chromatin organisation and maintenance of genomic stability may also play roles in checkpoint and other responses. Further screening, taking advantage of the types of phenotypes discovered in this screen, will be needed to identify these genes.