Genetics - Theses

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Type: PhD thesis × Start date: 2000 × End date: 2009 × Author: Verkade, Heather M. ×

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    Regulation of the G2
    Verkade, Heather M. (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.
  • Item
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    Regulation of the G2
    Verkade, Heather M. (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.