Earp, C.L.; (2008) Investigation of the essential role of S. cerevisiae Mec1. Doctoral thesis , University of London.

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Abstract

S. cerevisiae Mec1 is an essential, chromosome-based, signal transduction protein, which is involved in DNA replication, repair, and recombination. The Mec1-dependent checkpoint response to genotoxic stress has been widely investigated. However the essential role, which is believed to be to upregulate dNTP synthesis for DNA replication, remains less well understood. I have used two independent approaches to investigate the essential role of MEC1 further. Firstly, the possibility that Mec 1 controls local dNTP synthesis at the replication fork was examined. It is known that in the absence of Mec1 function, chromosome breakage occurs in specific regions of the genome called "replication slow zones". Given the importance of Mec 1 in upregulating dNTP synthesis, this phenotype could be explained by an essential requirement for Mec1 to couple dNTP synthesis with replication fork progression in these regions. This possibility was examined using chromatin immunoprecipitation to look for association of Mec1 with the DNA during S-phase. Although Mec1 recruitment to the DNA in response to replication stress was observed, no recruitment was seen during unchallenged S-phase. Secondly, a novel multicopy suppressor of the lethality of the temperature sensitive mec1 alleles, mec1-4 and mec1-40, was characterised. This suppressor, GIS2, encodes a retroviral-like zinc-finger protein, homologous to mammalian CNBP. GIS2 has been previously suggested to be a negative regulator of the RAS-cAMP-PKA signalling pathway, which is important for coupling growth and cell cycle progression. Therefore, other conditions expected to downregulate PKA activity were tested for their ability to suppress lethality of mec1-4. As predicted, both deletion of RAS2 and growth on alternative carbon sources can suppress the lethality of mec1-4. The only known mechanism of suppressing the loss of MEC1 function is to increase dNTP levels. However, increased GIS2 expression did not have any significant effects on the known mechanisms of upregulating dNTP synthesis. Overall, I describe a novel genetic interaction between mec1-4 and GIS2. Possible mechanisms for this interaction are also discussed.

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