Type
Text
Type
Thesis
Advisor
Advisors: Hollingsworth, Nancy M.; Futcher, Bruce
Date
2017-12-01
Keywords
Molecular biology
Department
Department of Biochemistry and Cell Biology | Thesis
Language
en
Source
This work is sponsored by the Stony Brook University Graduate School in compliance with the requirements for completion of degree.
Identifier
http://hdl.handle.net/11401/78301
Publisher
The Graduate School, Stony Brook University: Stony Brook, NY.
Format
application/pdf
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. | 73 pages
Recommended Citation
Gaglione, Robert, "Negative charges at Mek1 kinase consensus sites downregulate Ndt80 activity during budding yeast meiotic prophase without affecting protein stability" (2017). Stony Brook Theses and Dissertations Collection, 2006-2020 (closed to submissions). 3795.
https://commons.library.stonybrook.edu/stony-brook-theses-and-dissertations-collection/3795