Mechanisms of Meiosis
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Figure 1: Meiotic DNA break formation and repair. A Spo11-dependent DNA break formation initiates meiotic crossover repair and linkage between homologous chromosomes. B Non-random distribution of DNA break formation; chromosomes contain DSB "hot" and "cold regions. Data adapted from .[less]
Figure 1: Meiotic DNA break formation and repair. A Spo11-dependent DNA break formation initiates meiotic crossover repair and linkage between homologous chromosomes. B Non-random distribution of DNA break formation; chromosomes contain DSB "hot" and "cold regions. Data adapted from .
Since its inception in 2012, a central focus of the Vader group has been on understanding meiosis, the specialized cell division that is required for sexual reproduction. We are using the unicellular eukaryote Saccharomyces cerevisiae (i.e. budding yeast) to study how the meiotic cell division program allows the faithful generation of haploid gametes from a diploid progenitor cell. The meiotic program follows the same basic cell cycle logic as the mitotic cell cycle program, with the additional of several meiosis-specific events that allow the development of haploid gametes. A key meiosis-specific innovation is the segregation of homologous chromosomes during the first meiotic chromosome segregation event (i.e. during meiosis I). In order to allow for a faithful assortment of homologous chromosomes in meiosis I, initially unpaired chromosomes need to be paired and physically linked together [1, 2].
Homologue linkage occurs during a gap phase in between meiosis S-phase and meiosis I generally referred to as G2/prophase. During this phase meiotic progenitor cells use chromosome fragmentation and repair to link homologous chromosomes. Meiotic chromosome fragmentation is initiated by the formation of hundreds of DNA double strand breaks (DSBs) throughout the genome. Meiotic DSBs are generated by the topoisomerase-like protein Spo11, which works in concert with additional proteins in what has been dubbed the "meiotic DSB machinery" (Figure 1) . After DSB formation, repair of these lesions is achieved using homologous recombination. Meiotic homologous recombination is biased towards using the sequences present on the homologous chromosome instead of the sister chromatids. This bias towards interhomologous repair in combination with a specific resolution of DNA repair intermediate structures will often yield crossover repair products, in which flanking sequences between homologous chromosomes are exchanged. In combination with established cohesion between sister chromatids (provided by the cohesin complex, which is laid down in S-phase) crossovers provide the physical linkages between homologues (Figure 1a).
Spo11 generates non-random DSB patterns, which means that certain regions in the genome are more likely to experience a DSB as compared to others (Figure 1b). In addition, where an eventual crossover (and thus homologue linkage) is created depends on the choice between different possible DBS repair pathways. Thus, the eventual meiotic crossover landscape is shaped by several factors . DSB formation and crossover formation critically endanger specific regions in the genome. For example, DSBs that are formed within repetitive DNA elements are prone to undergo non-allelic homologous recombination (NAHR), an event that can trigger genome destabilization . In addition, it has been demonstrated that crossovers that are placed in the vicinity of centromeric sequences (i.e. within pericentromeres) predispose to meiotic chromosome non-disjunction and the consequential development of aneuploid gametes. For example, in humans, centromere-proximal crossovers are strongly correlated with the incidence of Trisomy 21, the genetic defect underlying Down's syndrome .
Dna break formation and repetitive dna
A DSB within repetitive sequences is a dangerous event because the presence of multiple identical repair templates increases the likelihood of non-allelic homologous recombination (NAHR). NAHR leads to loss or gain of genomic information. meiotic NAHR is associated with congenital genetic disorders in humans, whereas somatic NAHR can contribute to tumorigenesis. In many organisms, arrays of repetitive sequences are protected against meiotic DSB formation, often through the formation of specialized chromatin. We study how meiotic NAHR is minimized through the generation of local cold DSB regions, and use the repetitive ribosomal DNA (rDNA) array of budding yeast as a model locus to this. Understanding how NAHR is minimized at the rDNA in meiosis will not only increase our understanding of meiotic DNA formation and regulation, it might also yield novel insight into the general control of homologous recombination.
Meiotic DSBs are repressed in the yeast rDNA array, but we recently found that the outermost sequences of the rDNA are particularly vulnerable for DSB formation and NAHR. Counteracting this vulnerability requires specialized protection mediated by the conserved AAA+-ATPase Pch2 together with the origin recognition complex component Orc1. Interestingly, the rDNA-associated chromatin, although required for DSB suppression across the entire array, promotes DSB formation within array borders. Thus, our work has defined the borders of repetitive arrays as fragile sites during DSB formation that require specialized protection against meiotic DSB formation. We are currently using whole genome analysis combined with cell biological and biochemical methods to define how Pch2 collaborates with Orc1 to influence the meiotic DSB landscape. In addition, we are investigating how the rDNA-associated Sir2-histone deacetylase influences the formation of DNA breaks.
Dna break formation and recombination in the vicinity of centromeres
Centromeres are the genetic elements within chromosomes that direct the assembly of the kinetochore. As such, centromeres are essential for proper chromosome segregation during mitosis and meiosis. Several studies have shown a strong correlation between meiotic homologous recombination close to centromeres (i.e. within pericentromeric regions) and chromosome missegregation. For example, a significant percentage of Trisomy 21 cases (the chromosomal condition that causes of Down Syndrome) is associated with parental pericentromeric recombination in meiosis. In addition, it has been demonstrated that homologous recombination is normally repressed close to centromeres. Using budding yeast, we aim to unravel how DSB formation and homologous recombination are controlled at centromeres. We recently found that centromeres play an active role in determining DSB formation during meiosis, and we are currently defining how the centromeres and its associated kinetochore proteins influence this process.
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