The interplay between chromosome structure and meiotic integrity
Sammanfattning: Sexually reproducing organisms employ a specialized cell division called meiosis to form unique, haploid gametes from a diploid precursor cell. Upon fertilization, two opposite-sex gametes fuse to create a zygote that will develop into the offspring. Fundamental to meiosis is the formation and repair of programmed DNA double-strand breaks (DSBs) during prophase. These DSBs are repaired using the homologous chromosome (homolog) as a template for homologous recombination, which creates linkages between the homologs that are essential for faithful segregation. Following prophase, two successive rounds of chromosome segregation ensue. The first, meiosis I (MI), segregates homologs and the second, meiosis II (MII), segregates sister chromatids. Recombination is dependent on chromosome structure, and events that alter the DNA landscape will impact meiotic fidelity and, in turn, genomic integrity in gametes and future offspring. Thus understanding the relationship between chromosome structure and meiosis can help gain insight into human fertility and underlying causes of genetic disorders. It was the goal of this thesis to investigate factors known to regulate chromosome structure and determine their influence on meiosis in the budding yeast Saccharomyces cerevisiae and mice. In addition, we tested a novel method to search for new mammalian meiotic factors that may impact fertility. In Paper I, we identified a role for the conserved Structural Maintenance of Chromosomes (SMC) 5/6 complex during meiotic recombination in budding yeast. smc5/6 complex mutants experienced a DSB-dependent segregation block, suggesting that the defect was caused by recombination. Consistent with this notion, Smc6-deficient cells accumulated high levels of recombination intermediates, particularly between sister chromatids, which is normally not seen in the wild type. Return-to-function studies indicated that the Smc5/6 complex was most crucial during resolution of recombination intermediates. These results suggest that the Smc5/6 complex works primarily in the resolution of recombination structures formed outside of homolog-directed pathways during meiosis. We characterized a role for DNA topoisomerases Top2 and Top3 during meiosis in S. cerevisiae by using meiosis-specific mutants in Paper II. Cells deficient for either Top2 or Top3 experienced a segregation block. While top3 cells were rescued completely by removing recombination, the top2 mutant was only partially rescued. This suggests that Top3 mainly functions during meiotic recombination. In contrast, the data indicates that Top2 has a role outside of recombination. In line with this idea, some of the segregation defects in cells lacking Top2 seemed to arise from break-independent sister entanglements. Since Top2 is known to be important in resolving sister chromatid intertwinings during mitosis to facilitate proper segregation, it is likely that it plays a similar role during meiosis. The CCCTC-binding factor (CCTF) is an architectural protein essential for proper genome structure and function in higher eukaryotes. In Paper III, we created a testes- specific ctcf mouse mutant strain (cctf-cKO) in order to study the function of CTCF during gamete (sperm in males) formation in mice. CTCF-deficient mice completed meiosis and sperm specialization without any major abnormalities, though mice were infertile and had low sperm counts. Sperm from the ctcf-cKO had chromatin compaction defects, most likely due to lack of sperm-specific compaction factors. These findings indicate that CTCF is essential for proper chromatin organization during spermiogenesis and suggest that infertility in ctcf-cKO mice was a result of the chromatin defects in the sperm. Using a method called phylogenetic profiling in Paper IV, we showed that new meiotic factors can be discovered by clustering proteins according to their function.
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