Claeys Bouuaert, CorentinChristoph, KathleenKathleenChristoph2025-05-142025-05-142025-05-142022https://hdl.handle.net/2078.2/26297In the meiotic cells, a mechanism called the homologous recombination is implied in the genetic diversity, and is initiated by the formation of a DNA double strand break (DSB). Due to the particularly dangerous character of this phenomenon for the cell whose the DNA must be repaired to conserve the genome integrity, this mechanism is highly spatially and temporally regulated. In yeast, ten proteins are involved in the formation, the regulation and the repair of these DSBs. The protein Spo11, an homolog of the topoisomerase TopoVI, is the catalytic subunit which produces this break. In order to develop a system to measure the Spo11-mediated DSBs formation, this project allows to make two headways. First, an heterologous expression vector for the core complex proteins of Saccharomyces cerevisiae has been developed allowing their expression in Escherichia coli from an operon. Another strategy to express these proteins under the control of individual promoters has been also developed, but needs to be finished. Second, several strategies have been used to develop a SOS reporter system based on the SOS response. In the beginning, plasmids containing a LacZ gene under the control of a SOS-inducible promoter have been constructed. The expressed phenotypes of these plasmids and of another plasmid that contains a promoter not SOS-inducible indicate that this reporter system is not specific enough. Therefore, two strategies have been started to optimize it: either by insertion of terminators in the DNA sequence to avoid transcription leak through phenomenon, or by insertion of the reporter system in the chromosome to avoid transcription by mistake. Afterwards, a SOS reporter strain developed in another study has been also used. The results obtained by using this strain seem indicate some incompatibility between the SOS response and some antibiotics. By using these expression vectors for the core complex and these reporter systems, the identification of Spo11 hyperactive mutants could become possible.Spo11core complexDNA double strand breakDSBSaccharomyces cerevisiaetext::thesis::master thesisthesis:33851