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Investigating the Role of the Srs2 DNA Helicase during Meiosis in S. cerevisiae

Hunt, Laura (2018) Investigating the Role of the Srs2 DNA Helicase during Meiosis in S. cerevisiae. PhD thesis, University of Sheffield.

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During cell division, duplicated chromosomes must be segregated faithfully to prevent aneuploidy in daughter cells. In meiosis, there are two rounds of division following a single round of DNA replication. In the first meiotic division, crossovers formed between homologous chromosomes, via homologous recombination, ensure correct DNA segregation. Homologous recombination is intitated by DNA double-strand breaks (DSBs), which are processed to form single-stranded DNA that can invade donor duplexes to effect repair. In yeast, formation of nucleoprotein-filaments (NPFs) by RecA homologues Rad51 and Dmc1 promotes strand invasion into sister chromatids or homologous chromosomes, respectively. I investigated the meiotic role of Srs2, a multi-functional DNA helicase that is thought to regulate mitotic strand invasion via promotion of Synthesis-Dependent Strand Annealing to prevent hyper-recombination. To investigate meiotic phenotypes of srs2 strains, including deficient meiotic progression and reduced spore viability, I analysed nuclear and spindle pole body (SPB) division of spread chromatin. I found a significant increase in single nucleus cells with divided SPBs in srs2, suggesting cells are attempting to progress into second meiosis despite the nucleus failing to divide. Using strains with integrated TetO repeats and TetR-GFP to observe division at a single chromosome level, I conclude that homologues and sister chromatids are moving apart even when the nucleus fails to divide. Immunofluorescence of Rad51, in srs2 cells, revealed bright Rad51 foci appearing as aggregates under standard microscopy, which colocalise with RPA. These are dependent on SPO11, NDT80 and Rad51 strand invasion activity, but independent of MEK1 and SAE2, suggesting the meiotic phenotype is related to DSB formation and pachytene exit but independent of DSB resection or inter-homologue strand invasion. Interestingly, a partial rescue is observed when MRX complex formation is hindered. To determine whether Rad51 aggregation occurs at known recombination hotspots, I have prepared strains for ChIPSeq to analyse any alterations in the distribution of Rad51 along the DNA. Finally, the implications for the role of Srs2 during meiosis raised by these novel observations will be discussed.

Item Type: Thesis (PhD)
Academic Units: The University of Sheffield > Faculty of Science (Sheffield) > Molecular Biology and Biotechnology (Sheffield)
Identification Number/EthosID: uk.bl.ethos.739883
Depositing User: Dr Laura Hunt
Date Deposited: 05 Apr 2018 15:08
Last Modified: 28 Jan 2019 15:03
URI: http://etheses.whiterose.ac.uk/id/eprint/19942

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