Interactions between RMM complex proteins and their effect on DSB formation

Details

Supervisors

Claeys Bouuaert, Corentin ; Aldaccache, Dima

Faculty

Faculté des sciences

Degree label

Master [120] en biochimie et biologie moléculaire et cellulaire, à finalité approfondie

Abstract

The regulation of double-strand break (DSB) formation during meiosis is crucial for ensuring genomic stability and successful recombination. This study investigates the role of the Rec114 protein in DSB formation and its interactions with Mer2, another essential component of the RMM complex. Using mutagenesis, protein interaction assays, and genetic approaches, we dissected the molecular interface between Rec114 and Mer2. Key residues in the Mer2 SSM1 domain (notably I55 and W58) and the Rec114 PH domain (such as I34) were identified as critical for their interaction. From the hypothesis that the coiled-coil structure of Mer2 could cause steric clashes and may need to partially dissociate to enable Rec114 binding. A Mer2 mutant destabilizing the coiled-coil was designed to find insights into its interaction dynamics with Rec114. The mutant highlighted a potential but subtle influence on Rec114 binding, with ionic strength-dependent effects observed in pull-down assays. This suggests that Mer2's structural dynamics may contribute to the regulation of its interaction with Rec114, though further validation using advanced structural techniques is required. Additionally, this thesis investigated the effect of Rec114 overexpression on DSB levels. Plasmid-based and genomic integration systems confirmed high levels of Rec114 expression under strong promoters; however, these levels did not correlate with significant changes in DSB frequency or cellular viability, as assessed through Return to Growth assays and tetrad dissections. The implementation of Southern blot analysis is necessary to definitively assess the effect of Rec114 overexpression on DSB formation. Together, these findings provide insights into the molecular interactions and regulatory mechanisms governing meiotic DSB formation, laying a foundation for future studies to unravel the intricate dynamics of the RMM complex and its role in recombination.