Biochemical Reconstitution of the Budding Yeast Meiotic Chromosomal Axis

Abstract

Dissertation gesperrt bis zum 30.06.2028!

The production of gametes requires a specialised cell division to divide the genome, thus enabling the faithful transmission of genetic material from one generation to the next. This process is known as meiosis, the hallmark of which is meiotic recombination. A striking feature here is the physical exchange of DNA between homologous chromosomes, also known as crossing over, which is essential for ensuring that chromosomes segregate accurately. Moreover, genetic exchange between homologous chromosomes promotes species diversity – contributing to the overall evolution of Eukarya. It begins with the highly regulated formation of double-stranded DNA breaks (DSBs) throughout the genome, which take place within the context of a DNA-loop and proteinaceous axis structure. Crucially, a sub-set of these breaks must be repaired using the homologous chromosome as a template, as this is a pre-requisite for crossover formation. In Saccharomyces cerevisiae, this inter-homolog biased repair is ensured by a meiosis-specific protein complex: Hop1-Red1-Mek1 (HRM). Hop1 and Red1 form the structural basis of the proteinaceous axis, whereas Mek1 kinase is involved in both mediating inter-homolog bias as well as meiotic progression. Considerable in vivo efforts have been conducted to understand the functions of these proteins in meiosis, however there is limited knowledge about how HRM form a complex. Moreover, the assembly mechanism of the budding yeast axis itself is still poorly understood on the molecular stage. To extend our understanding of the molecular determinants required for the assembly of the HRM complex, I utilised an in vitro structural biochemistry approach. I investigated how the HRM complex is established and activated by designing a series of protein truncations and mutants, and revealed previously unrecognised roles for the N-terminus of Red1 in its interaction with Hop1 and Mek1. To characterise the enzymatic properties of Mek1, I recapitulated the meiotic conditions required for kinase activation in vitro. Finally, I explored the potential roles that the axis plays in the recruitment of key DSB machinery and cross-over formation proteins. Through this work, I propose a novel model whereby the HRM complex is potentially assembled and primed temporally early on to mediate inter-homolog bias at the onset of meiotic DSBs. The results presented here address the long-standing question regarding how Mek1 is recruited to axial sites and provide insight into the importance of the meiotic chromosome axis in mediating multiple branches of meiotic recombination in budding yeast.