Sexual reproduction in eukaryotes involves the generation of haploid gametes (in humans, sperm and egg cells) in meiosis, followed by the fusion of two gametes to produce diploid offspring. In meiosis, homologous chromosomes recognize one another and become physically linked through a modified homologous recombination DNA repair pathway, and the resulting crossovers enable accurate homolog segregation in the meiosis I division to reduce ploidy. In most eukaryotes including humans, chromosomes are organized as an array of chromatin loops by a highly conserved structure called the chromosome axis. The chromosome axis also recruits and controls DNA cleavage and recombination factors to mediate the formation of crossovers, and is remodeled after crossover formation in a key feedback pathway controlling recombination levels. Here, we propose to combine biochemistry, macromolecular structure, and genetics in both S. cerevisiae and the mouse to determine how the chromosome axis assembles, organizes chromosomes, and mediates crossover formation. We will first determine the structures of S. cerevisiae Red1 and mammalian SYCP2:SYCP3, functionally-related chromosome axis ?foundation? proteins that we have found share a conserved domain structure and propensity to self-assemble into filaments. We will next determine how these proteins interact with meiotic cohesin complexes, to understand the structural basis for axis-mediated chromosome organization. Next, we will dissect the network of interactions mediated by S. cerevisiae Hop1, a member of the conserved HORMAD family of axis proteins and a master regulator of meiotic recombination, and study how this interaction network changes during as meiotic prophase progresses. Hop1's eventual removal from the chromosome axis, an important feedback pathway controlling recombination levels, is mediated by the AAA+ ATPase Pch2. We will test our hypothesis that Pch2 directly recognizes a specific Hop1 conformation and partially unfolds its HORMA domain to mediate its removal from the axis. Finally, we will examine the structures, DNA binding specificity, and interactions of two meiosis- specific protein complexes, Msh4:Msh5 and Zip2:Zip4:Spo16, to learn how they stabilize specific DNA recombination intermediates and coordinate crossover formation with chromosome axis morphology changes. Overall, the work proposed here will result in a comprehensive molecular picture of how the chromosome axis assembles, coordinates crossover formation, and is then disassembled as recombination proceeds. Understanding the molecular mechanisms of the chromosome axis and associated factors is highly relevant to human health, as errors in meiotic chromosome segregation are a principal cause of miscarriage in humans, and are the source of ?aneuploidy disorders? like Down syndrome and Turner syndrome. Moreover, many cancer types show mis-expression of meiotic chromosome axis proteins, including TRIP13, HORMAD1, and SYCP2. A better understanding of these proteins' mechanisms in their native environment will be critical to determine how their mis-expression might lead to genome instability and cancer.
We are exploring the fundamental mechanisms of how chromosomes are recombined and divided during sexual reproduction, where errors can result in aneuploidy (gain or loss of chromosomes) and consequent miscarriage (up to 15-20% of pregnancies) or developmental disorders like Down syndrome (1 in 1,000 live births), Turner syndrome, and others. Many of the proteins we study are also mistakenly expressed in cancer cells, and while their contributions to cancer are not known, they likely impact diverse DNA repair pathways and chromosome segregation mechanisms to cause genome damage and aneuploidy in these cells. By determining the structures and biochemical mechanisms of important molecular machines involved in meiosis, we will provide foundational knowledge for understanding their biological roles in both human reproduction and cancer.
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