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Negative charges at Mek1 kinase consensus sites downregulate Ndt80 activity during budding yeast meiotic prophase without affecting protein stability

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dc.contributor.advisor Hollingsworth, Nancy M en_US
dc.contributor.author Gaglione, Robert en_US
dc.contributor.other Department of Biochemistry and Cell Biology en_US
dc.date.accessioned 2018-07-03T17:22:59Z
dc.date.available 2018-07-03T17:22:59Z
dc.date.issued 2017-12-01 en_US
dc.identifier Gaglione_grad.sunysb_0771M_13553.pdf en_US
dc.identifier.uri http://hdl.handle.net/11401/78301 en_US
dc.description 73 pgs en_US
dc.description.abstract Meiotic recombination promotes the formation of crossovers between homologous chromosomes to ensure their proper segregation in Meiosis I. Crossovers are formed by the repair of programmed double strand breaks (DSBs) during meiotic prophase. In budding yeast, the meiotic recombination checkpoint delays meiotic progression until all DSBs have been repaired and is under the control of the meiosis-specific Mek1 kinase. The meiosis-specific transcription factor Ndt80 activates the transcription of hundreds of genes, including those required for prophase exit and the meiotic divisions. Transcription of NDT80 occurs in two stages, mediated first by Ime1 and then by Ndt80 itself. Ime1-transcribed Ndt80 is negatively regulated by the checkpoint, preventing the expression of NDT80 and its downstream targets. Mutated strains which arrest due to unresolved DSBs accumulate inactive Ndt80 in the cytoplasm. Ndt80 contains six putative Mek1 consensus sequences within its DNA-binding domain. While preventing phosphorylation at these sites using alanine substitutions (ndt80-6A) does not affect Ndt80 activity, aspartic acid substitutions (ndt80-6D), which may mimic the negative charge of phosphorylation, make Ndt80 constitutively inactive. Immunoblot analyses showed that ndt80-6D prevents accumulation of Ndt80-dependent gene products, including Ndt80 itself. When the NDT80 gene was put under the control of an inducible promoter, Ndt80-6D protein levels were equivalent to wild-type, although no downstream targets were detected. This result shows that negative charges do not affect Ndt80 protein stability, but instead prevent the accumulation in Ndt80 protein that results from Ndt80-mediated transcription of NDT80. In addition, a fluorescence-based system was developed to allow for the visualization of Ndt80 localization in live cells. en_US
dc.description.sponsorship This work is sponsored by the Stony Brook University Graduate School in compliance with the requirements for completion of degree. en_US
dc.format Monograph en_US
dc.format.medium Electronic Resource en_US
dc.language.iso en_US en_US
dc.subject.lcsh Molecular biology en_US
dc.title Negative charges at Mek1 kinase consensus sites downregulate Ndt80 activity during budding yeast meiotic prophase without affecting protein stability en_US
dc.type Thesis en_US
dc.mimetype Application/PDF en_US
dc.embargo.release 2018-08-13 en_US
dc.embargo.period 4 en_US
dc.contributor.committeemember Futcher, Bruc en_US

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