Study of the conservation of the structure and the function of S. cerevisiae Rec114-Mei4-Mer2 proteins during evolution

Details

Supervisors

Claeys Bouuaert, Corentin ; Morsomme, Pierre

Faculty

Faculté des bioingénieurs

Degree label

Master [120] : bioingénieur en chimie et bioindustries, à finalité spécialisée

Abstract

In prophase I of meiosis, the phenomenon of genetic recombination takes place, which can lead to the formation of crossovers between two homologous chromosomes. This phenomenon is essential for the correct segregation of homologous chromosomes at the end of anaphase I of meiosis. Genetic recombination is initiated by a DNA double-strand break mediated, in S. cerevisiae, by the Spo11 protein. However, this phenomenon also requires the presence of 9 other proteins divided into three complexes: The core complex (which contains the Spo11 catalytic unit), the MRX complex and the RMM complex (composed of Rec114, Mei4 and Mer2). It has been shown in vivo and in vitro that the proteins of the RMM complex form condensates in the presence of DNA. This phenomenon is referred to as phase separation. It is hypothesized that this complex acts as a platform that recruits the 7 other essential proteins to the site where DNA double-strand breaks occur. The aim of this master thesis is to study the conservation of the structure and DNA-binding function of the S. cerevisiae RMM complex orthologs throughout evolution. As these proteins cannot be purified together, the study is divided into two distinct parts: A part on the Rec114-Mei4 orthologs where the complexes from mice (REC114-MEI4), S. pombe (Rec7-Rec24), A. thaliana (PHS1-PRD2) and Z. mays (PHS1-MPS1) will be studied and another on Mer2 orthologs where the protein from mouse (IHO1), S. pombe (Rec15), A. thaliana (PRD3), Z. mays (PAIR1) and S. macrospora (ASY2) will be studied. Despite the large difference in amino acid sequences between these orthologous proteins, the structure predicted by AlphaFold is quite similar. These models of the complex formed by orthologs of Rec114 and Mei4, with a 2:1 stoichiometry respectively, will be verified by mutagenesis followed by a Pulldown in order to identify the amino acids important in the protein interactions between the C-terminal part of Rec114 and the N-terminal part of Mei4. Concerning the Mer2 orthologs, only the structure of the parts of the proteins forming a coiled-coil domain will be studied by SEC-MALS, SAXS and XL-MS, to see whether the homotetrameric and parallel structure of Mer2 is conserved. In order to study the DNA-binding function of these proteins, EMSAs were carried out on the minimal complexes and coiled-coil domains of the proteins studied. In S. cerevisiae, these proteins bind DNA in vitro and the conservation of this activity is studied in this work.