| Notes: | Advisor: Douglas K. Bishop.
Thesis (Ph.D.)--The University of Chicago, Division of the Biological Sciences, and The Pritzker School of Medicine, Committee on Genetics, 2013.
Dissertation Abstracts International, Volume: 74-07(E), Section: B.
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| Summary: | Interhomolog crossover recombination is required for proper segregation of homologous chromosome pairs during meiosis I. Interhomolog crossovers are established by a specific recombination mechanism that avoids the use of sister chromatids as recombination partners. The two strand exchange proteins, Rad51 and Dmc1, are important for the interhomolog recombination preference. The specific functions of these two strand exchange proteins during meiosis are unknown.
In order to study the two strand exchange proteins, separation of function mutants were created that allow for filament formation but not strand invasion. The Rad51 separation of function mutant had wild-type levels of meiotic recombination and wild-type interhomolog partner bias; this is in contrast to the rad51, which has lowered recombination levels and intersister bias. Further, the Rad51 mutant promoted loading of Dmc1 filaments in contrast to the rad51Δ mutant and in vitro analysis suggests that Rad51 promotes the strand exchange activity of Dmc1. In stark contrast, the Dmc1 separation of function mutant arrested similar to a dmc1Δ mutant.
Hed1 was discovered as an inhibitor of Rad51 activity during meiosis. In order to study the function of Dmc1 in more detail, meiosis was analyzed in a dmc1Δ hed1Δ mutant, which overcomes the meiotic arrest of dmc1Δ mutants. This mutant allowed for the analysis of meiosis in a background in which Rad51 was carrying out strand exchange in the absence of Dmc1, allowing for the identification of Dmc1-specific roles during meiotic recombination. This analysis has revealed that dmc1Δ hed1Δ mutants are defective in interhomolog bias.
In addition to mechanisms that ensure interhomolog recombination, there are mechanisms that control the number and positions of crossovers. Analysis of tetrads in dmc1Δ hed1Δ mutants shows that they are defective in crossover control. Despite severe defects in partner choice and crossover control, dmc1Δ hed1Δ mutants have near normal recombination rates suggesting a compensation mechanism for reduced interhomolog events. Consistent with this, dmc1Δ hed1Δ cells show a higher percentage of interhomolog interactions maturing to crossovers rather than non-crossovers relative to wild-type cells.
In addition to defects in recombination, dmc1Δ mutants have defective assembly of the synaptonemal complex, a meiosis specific structure that forms between homolog pairs. To determine if this defect is due to the absence of recombination in dmc1Δ mutants or due to a specific requirement for Dmc1, synaptonemal complex formation was monitored in dmc1Δ hed1Δ mutants. Despite a partial rescue in synaptonemal complex formation in dmc1Δ hed1Δ mutants, these mutants show defects in the abundance of synaptonemal complex proteins. Thus, Dmc1 is required for retention or recruitment of synaptonemal complex proteins.
The separation of function mutants demonstrate that Rad51's strand exchange activity is dispensable for wild-type meiosis, whereas that of Dmc1 is indispensable for wild-type meiosis. Further, Rad51 acts as an accessory factor for Dmc1, in part by promoting Dmc1 filament formation. Analysis of the dmc1Δ hed1Δ mutant suggests that Rad51 cannot substitute for Dmc1 in interhomolog bias, crossover control, and synaptonemal complex formation. The inability of Rad51 to substitute for Dmc1 is likely due to the unique set of accessory factors used by Dmc1, such as Mnd1. Coordination with meiosis specific accessory factors, including Rad51, likely plays a key role in interhomolog bias during meiosis. These data taken together provide support for a model in which an inhibited version of Rad51 is required to direct the activity of Dmc1 toward the homolog with Mei5-Sei3 acting as a linker between the two strand exchange proteins. This is likely in coordination with checkpoint proteins, which together allow for a scaffold directing Dmc1 toward the homolog rather than the sister.
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