Biochemical Dissection of Mer2 in Meiotic DNA Double-Strand Break Formation

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Dokumentart: PhDThesis
Date: 2024-05-01
Language: English
Faculty: 7 Mathematisch-Naturwissenschaftliche Fakultät
Department: Biologie
Advisor: Weir, John (Dr.)
Day of Oral Examination: 2022-06-02
DDC Classifikation: 500 - Natural sciences and mathematics
570 - Life sciences; biology
Other Keywords: Biochemie
in vitro
double-strand breaks
in vitro
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Meiotic cell division is a critical step in sexual reproduction that leads to the formation of haploid gametes from a diploid cell. During genome reduction, the homologous chromosomes are segregated into daughter cells. To avoid missegregation, the chromosomes have to be physically linked via homologous recombination. This linkage is enabled by the repair of programmed double-stranded DNA breaks (DSBs) using the homologous chromosome as a repair template rather than the sister chromatid. Introducing DSBs in the genome is precarious and has to be strictly controlled to avoid irreparable damage to the organism itself and its offspring. The break site is localized by an H3K4me3 mark on nucleosomes, which is recognized by the PHD domain-containing protein Spp1. Spp1 interacts with the protein Mer2, which connects the break site with the break machinery, localized in the proximity of the chromosomal axis, via its binding to the axial proteins Hop1 and Red1. These interactions must take place at the right position in the chromosome at the right time in the cell cycle to form breaks. Although the mechanism of DSB control has been studied for many years, its underlying molecular details remain to be deciphered. To reveal the essential molecular elements involved in this process, I used Saccharomyces cerevisiae as a model organism and adopted an in vitro approach combining biochemical and structural methods on purified recombinant proteins. The DSB control was first explored by interaction experiments with Spp1, a nucleosome mark reader, and Mer2, a chromosomal axis interactor. The results demonstrate that they form a constitutive complex with 2:4 stoichiometry at low nanomolar affinity. Dimerization of Spp1 by Mer2 strengthens its interaction with the nucleosome. Moreover, not only Spp1 but also Mer2 is a novel nucleosome binder, forming a stable complex with recombinant nucleosomes in solution. The interaction of Mer2 with nucleosomes provides additional stability to the assembly, where Spp1 provides the specificity of the interaction and Mer2 the strength. Once the future DNA break site is localized via its interaction with Spp1 and Mer2, it must interact with the chromosomal axis formed by Hop1-Red1 and cohesin, where the break machinery is. My data reveal that the conserved C-terminal region of Mer2 specifically interacts with an axis-bound Hop1 to ensure that breaks are made only when the chromosomal axis is properly formed. An additional level of control is provided by the conserved N-terminal region of Mer2, which is crucial for DSB formation. The N-terminal region establishes a previously undescribed connection with protein Mre11, which is responsible for resection of the DSBs, thus demonstrating that the factors both to create DNA break and repair it have to be in place before the break occurs. Collectively, these findings reveal that Mer2 serves as an interaction platform for proteins involved in the control of DSBs, rendering it an essential component of proper DSB formation and resection. Moreover, they provide insights into the molecular details of DSB control and serve as a foundation for further studies of meiotic DSB formation. Illuminating previously unnoticed levels of DSB control significantly extends our understanding of the process of homologous recombination and, ultimately, meiosis as a whole.

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