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Title: The repair of DSBs catalyzed by VMA1 derived endonuclease by homologous recombination during meiosis
Author: Medhi, Darpan K.
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2013
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Homologous recombination (HR) during meiosis is initiated by programmed DNA double strand breaks (DSBs). Some of these DSBs are repaired to give crossovers (COs), which connect maternal and paternal homologous chromosomes and thus ensure proper segregation during meiosis I. In contrast, HR in mitotic cells forms mostly noncrossovers (NCOs); this prevents deleterious genome rearrangement and loss of heterozygosity. Therefore, meiotic HR is regulated to enrich for COs, but much remains to be understood regarding the basis of this regulation. Meiotic cells express unique HR proteins, and these global factors might facilitate the distinct regulation of meiotic HR. In addition, meiosis-specific proteins localize to the chromosome axis, and these proteins interact with the meiotic Spo11 complex during DSB formation. Thus, the substrate for meiotic HR forms in a unique local chromatin context, and is then acted upon by unique global recombination factors. In order to better understand the balance between local and global influences, we studied the meiotic repair of DSBs formed by the VMA1-derived endonuclease (VDE). Repair of these breaks, which form independent of the meiotic chromosome axis, should still be influenced by global cell wide meiotic recombination proteins, but may or may not be influenced by the localized meiotic chromosome axis. We studied repair of two VDE DSBs: one located in a region that is "hot" for Spo11 DSBs and is enriched for axis proteins; and one in a Spo11 DSB "cold" region that is not enriched for axis proteins. VDE DSBs are repaired at both loci to produce NCOs in excess over COs, but more COs are formed at the hot-spot locus. The hot-spot also accumulated more joint molecules (JMs), which are the intermediates of CO formation. In addition, CO formation shows different resolvase dependence at the two loci. Hot-spot COs are Mlh3-dependent but largely independent of the "mitotic" Mus81-Mms4 structure-selective nuclease; whereas cold-spot COs are Mlh3-independent but show dependence on Mus81- Mms4. Finally, in spo11-Y135F cells lacking genome wide meiotic DSBs, VDEinitiated COs are reduced at both loci, which now display an identical NCO-CO ratio. This effect is partially attributable to the lack of pairing in these cells, as ectopic repair of VDE DSBs in SPO11 cells also have a similar NCO-CO ratio. Thus, COs in meiosis require a specific global recombination environment. COs are also influenced by factors that act locally, such that VDE initiated COs at a hot-spot and a cold-spot are reminiscent of meiosis and mitosis respectively.
Supervisor: Goldman, Alastair S. H. ; Lichten, Michael J. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available