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dc.contributor.advisorLahue, Bob
dc.contributor.authorConcannon, Claire
dc.date.accessioned2013-12-18T09:57:30Z
dc.date.available2014-10-10T15:07:21Z
dc.date.issued2013-09-23
dc.identifier.urihttp://hdl.handle.net/10379/3921
dc.description.abstractTrinucleotide repeat (TNR) expansions are the genetic cause of several inherited neurological diseases. Factors that affect the propensity of TNRs to expand include the characteristics of the TNR DNA, the chromatin environment, and the presence of cellular factors that enhance or inhibit expansions. TNRs that expand and their resulting diseases have a threshold, a TNR length range that demarcates stable and unstable alleles. Expansions that cross the threshold are the key disease-initiating mutations. The mechanisms by which TNRs expand are not well defined, especially for threshold-length expansions. A widely accepted model predicts that during DNA processing the TNR forms an abnormal secondary structure, which is then converted to an expansion. Certain proteins in the cell are thought to either inhibit or promote this process. This study investigates novel promoting factors of threshold-length TNR expansions using a selective genetic assay that allows quantification of expansion rates. Two model systems were employed, the yeast Saccharomyces cerevisiae and a human astrocytic cell line. In yeast, genes of interest were targeted for deletion by a PCR based method and the resulting expansion rate compared to wild type. In human cells, RNA interference was used to reduce the levels of the protein of interest and the resulting expansion frequency compared to a scrambled siRNA control. A previous blind screen for novel promoting factors of TNR expansions implicated the SEM1 gene. Investigation of this mutant revealed that the 26S proteasome is a promoting factor of TNR instability. Deletion of several 26S proteasome subunits, or chemical inhibition of the proteasome, suppressed TNR expansions in yeast. siRNA mediated knockdown of human 26S proteasome subunits in an astrocytic cell line also suppressed expansions. Suppression of expansions in both yeast and human cells was concurrent with a reduction in proteolytic activity, as determined by accumulation of polyubiquitinated proteins or by a proteasome activity assay. This provides evidence that protein proteolysis is an important mechanism for promoting repeat instability. Further results imply a role for the nucleotide excision repair (NER) pathway in promoting TNR expansions in yeast. Deletion of several NER factors involved in transcription-coupled and/or global genome NER stabilized TNR expansions. Analysis of proteasome/NER double mutant suggested that the proteasome and NER act through a common pathway to facilitate expansions.en_US
dc.rightsAttribution-NonCommercial-NoDerivs 3.0 Ireland
dc.rights.urihttps://creativecommons.org/licenses/by-nc-nd/3.0/ie/
dc.subject26S proteasomeen_US
dc.subjectTrinucleotide repeat expansionen_US
dc.subjectNucleotide excision repairen_US
dc.subjectNeurological diseaseen_US
dc.subjectBiochemistryen_US
dc.titleIdentification of novel factors that promote trinucleotide repeat instabilityen_US
dc.typeThesisen_US
dc.contributor.funderCollege of Science, NUI Galwayen_US
dc.contributor.funderThomas Crawford Hayes Research Funden_US
dc.contributor.funderScience Foundation Irelanden_US
dc.local.noteExpansion of repetitive DNA underlies several neurological disorders in man. It has been found that some proteins within the cell can drive this expansion process. This work identifies and characterises several new proteins that promote expansions. Thus, it provides possible avenues for therapeutic interventions to help prevent these disease-causing mutations.en_US
dc.local.finalYesen_US
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Attribution-NonCommercial-NoDerivs 3.0 Ireland
Except where otherwise noted, this item's license is described as Attribution-NonCommercial-NoDerivs 3.0 Ireland