Genotype-phenotype analysis of the checkpoint kinase MEC1, the budding yeast homologue of human ATM/ ATR

Waskiewicz, Erik (2017) Genotype-phenotype analysis of the checkpoint kinase MEC1, the budding yeast homologue of human ATM/ ATR. PhD thesis, Prifysgol Bangor University.

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The protein kinases ATR and ATM, and their Saccharomyces cerevisiae counterparts Mec1 and Tel1, are the apical kinases of the DNA damage response, a cellular response to potentially lethal DNA damage from both endogenous and exogenous sources. ATR/ATMMec1/Tel1 are part of the PIKK (PI3-kinase-like kinases) family of protein kinases; PIKK proteins are very large and contain a conserved C-terminal kinase domain and a large number of N-terminal helical HEAT repeats, thought to be involved in target binding. Mutation of ATM leads to the developmental disorder Ataxia-Telangiectasia, and ATM and ATR have been found to be mutated in a large number of cancers. Although much is known about the role of ATM/ATR in the DNA damage response, very little is known about the structure-function relationship of the proteins. ATM/ATR can phosphorylate around 700 different targets, yet how they bind and how target specificity is controlled remains largely unknown. In this study I have identified 23 randomly mutated Mec1ATR alleles to assess which region of the protein is important for the response to different stresses; particularly hydroxyurea (HU), which causes replication stress, and methyl-methansulphonate (MMS), which causes DNA damage. There were no mutations identified at the very N-terminal region of Mec1, the region where Ddc2ATRIP binds, suggesting that mutations in this region may effect this essential interaction. The remaining mutations were spread over the length of the protein and many mutations were found in the kinase domain. The mutation E2130K in the kinase domain was found in two seperate strains. In most instances a sensitivity to a certain stress correlated with a loss of Mec1s ability to phosphorylate Rad53, a major downstream effector kinase. Interestingly, in some cases, combining two different mec1 alleles in the same strain rescued their respective phenotypes, indicating intramolecular complementation of the dimeric Mec1 kinase. A number of these alleles are also conserved in human ATM/ATR and have been shown to be mutated in cancer.

Item Type: Thesis (PhD)
Subjects: Degree Thesis
Departments: College of Health and Behavioural Sciences > School of Medical Sciences
Degree Thesis
Date Deposited: 13 Nov 2017 14:50
Last Modified: 13 Nov 2017 14:50
URI: http://e.bangor.ac.uk/id/eprint/10269
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