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Title: Monopolin complex fuses sister kinetochores specifically in meiosis I
Author: Plowman, Rebecca
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2020
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Meiosis is a specialised form of cell division, which halves the chromosome content of a cell. A single round of DNA replication is followed by two rounds of division. Meiosis utilises the key machinery of mitosis but with specific adaptations, particularly in the first division. In the first meiotic division, unlike mitosis or meiosis II, sister chromosomes segregate together. In budding yeast, the fusion of sister kinetochores allows them to attach to a single microtubule from one cell pole. The four-protein complex responsible for this fusion and therefore sister co-segregation in meiosis I is called monopolin. However, the mechanism by which monopolin acts is unclear. What is well understood about monopolin is its structure, it is a four-protein complex of two homodimers of Csm1 and two copies of the proteins Lrs4, Mam1 and the casein kinase, Hrr25. The complex forms a V-shaped structure, with a kinetochore binding region at each point of the V. This structure has led to the proposed mechanism of monopolin forming a mechanical bridge between the two kinetochores directly causing their fusion. There is, however, no direct evidence for this mechanism and many open questions remain, particularly as to how the bridging interaction would be regulated and the role of proteins, Mam1 and Hrr25, which do not form the structure of the V-shape. This project had several aims, firstly to dissect whether all components of the monopolin complex are required for monoorientation or if some only serve to target the complex to the kinetochore. By artificial tethering of monopolin to the kinetochore, I was able to demonstrate the requirement for a complete monopolin complex for the mono-orientation of sister chromatids. Secondly, I aimed to show the molecular basis for the interaction between monopolin and the kinetochore. This was done by following insight gained form the crystal structure of monopolin showing the interaction between Csm1 and its kinetochore receptor Dsn1, I generated mutants in Dsn1 that disrupt this interaction. Through predominantly assessment of cell viability, imaging of chromosome division in meiosis and chromatin immunoprecipitation of monopolin components I demonstrated that these sites within Dsn1 are essential for the localistion of monopolin to the kinetochore, sister chromatid mono-orientation and therefore meiosis. In addition, through the use of mass spectrometry I showed that two serines within this region of Dsn1 are phosphorylated in meiosis, and that they are good candidates for being a point of regulation for the interaction between monopolin and the kinetochore. Finally, although it is known that Hrr25 must be enzymatically active for mono-orientation of sister chromatids in meiosis, no in vivo targets of this phosphorylation have been described. I, therefore, utilized a mutant version of Hrr25 which is not recruited to the monopolin complex and mass spectrometry to identify possible targets of phosphorylation by Hrr25 at the meiotic kinetochore.
Supervisor: Marston, Adele ; Welburn, Julie Sponsor: Medical Research Council (MRC)
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: meiosis ; meiotic division ; monopolin complex ; kinetochores ; mono-orientation ; Hrr25