Meiotic Recombination is essential for the production of healthy gametes (egg and sperm in humans). Recombination creates physical connections between the maternal and paternal copies of each chromosome thereby making it possible for them to disjoin from one another during the first round of meiotic chromosome segregation. Errors in segregation result in birth defects, miscarriages, and infertility. This project is direced at the regulation of the key central enzyme in meiotic recombination Dmc1. Dmc1 is assembled into DNA-protein filaments at sites of DNA double strand breaks. These filaments have the ability to search intact chromatids for sequences that match the sequence bound within the filament and, having found such sequences, promote an exchange of DNA single strands to form regions of hybrid DNA involving a DNA single strand from the maternal chromosome and the complementary single DNA strand from the paternal chromosome. Assembly of Dmc1 filaments requires cooperation of accessory factors including a heterodimeric protein called Mei5-Sae3. In the previous grant cycle we showed that Dmc1's paralogue Rad51, which provides the only strand exchange activity in mitotic cells, is converted to an accessory factor that functions with Mei5-Sae3 to stimulate Dmc1's activity. In this work we will determine the mechanism through which Rad51 and Mei5-Sae3 cooperate using traditional biochemical approaches combined with single molecule analysis of filament structure using super-resolution light microscopy and other microscopic methods. Once Dmc1 filaments are assembled, a second accessory factor, Hop2-Mnd1 stimulates the activity of Dmc1 about 10 to 30 fold in vitro. Genetic experiments also show that Dmc1's activity requires Hop2-Mnd1. During the current funding period we showed that Hop2-Mnd1 specifically stimulates Dmc1 and not Rad51 leading to the proposal that Hop2-Mnd1 is responsible for the specialized function of Dmc1 in meiosis. We hypothesize that Hop2-Mnd1 regulates Dmc1 activity such that it preferentially promotes recombination with a homologous chromosome rather than a sister chromatid, i.e. such that recombination is between a maternal and paternal chromatid rather than between two maternal or two paternal chromatids. Here we proposed to test a specific molecular model for how this recombination partner bias is achieved via the regulated distribution of Hop2-Mnd1 on chromosomes. Finally, we will use super-high resolution light microscopy to characterize the structure of the meiotic recombination complex (recombinosome). In the previous funding period we found evidence that Dmc1 is forms complexes on both DNA ends created by a DNA double strand break which then separate from one another. We propose to determine how the two complexes are arranged with respect to the proteinaceous meiosis-specific chromosome scaffold structure called the axial/lateral element. These experiments will shed light the mechanism through which Dmc1 searches the genome for recombination partners.
Formation of healthy eggs and sperm requires accurate completion of meiosis, a specialized cell division program that accurately reduces the number of copies of each chromosome from 2 to 1; errors made in meiosis result in infertility, miscarriage, and birth defects. This project examines the mechanism of action of the key enzyme required to connect chromosome pairs in preparation for reductional division of chromosomes. The mechanism is very similar to a DNA repair mechanism that promotes genome stability making the work proposed relevant to the mechanism of cancer progression.
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