Ahmed Ezzat, Hassan and Price, Clive (2018) Characterisation of non-essential genes required for survival under conditions of DNA stress. PhD thesis, Lancaster University.
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Abstract
Genomic instability is a hallmark of cancer. Cancer progression depends on the development and amplification of mutations that alter the cellular response to threats to the genome. This can lead to DNA replication stress and the potential loss of genetic integrity of the newly formed cells. The study of the sophisticated systems of DNA replication has been an attractive area for research in human and cancer, yet, the results do not fully provide a clear understanding. This could be due to sheer complexity of these systems in Homo sapiens as some key elements in these processes have evolved to be multifunctional to several pathways rather than one. This project utilises fission yeast to map the interactions occurring in some of the most crucial pathways in both DNA replication and checkpoint monitoring. One particularly interesting protein that fits the multifunctional description, is TopBP1. Rad4 is the Schizsaccharomyces pombe (S. pombe) TopBP1 homologue that is essential for the initiation of DNA replication, DNA repair as well as the activation of DNA damage checkpoint signalling. We have modelled conditions of replication stress in the genetically tractable fission yeast, S. pombe using the hypomorphic rad4-116 allele. Synthetic genetic analysis was used to identify processes required for cell survival under conditions of DNA replication stress. With the aim of mapping the genetic interactions of rad4 and its mutant allele, rad4-116, several genes that could have an interaction with rad4 during replication stress have emerged as attractive. Interactions with genes involved in chromatin remodelling, such as hip1, and replication fork stalling resolution, such as mrc1, swi1 and swi3 were explored. Results from these studies will provide a clear view of the rad4 interactions, the association of Rad4 with the replisome which in turn should provide a more robust understanding of the pathways associated. Ultimately, this will serve as a platform for targeting these pathways for anti-cancer drug development.