Impact of active site mutations on SPO11 DNA binding & cleavage activity

Details

Supervisors

Claeys Bouuaert, Corentin

Faculty

Faculté des sciences

Degree label

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

Abstract

Meiosis is a specialized cellular process that generates haploid gametes from a diploid germ cell. A crucial feature of meiosis is the shuffling of genetic material, which ensures each gamete contains a unique blend of genes from both parents, playing a vital role in sexual reproduction. This genetic shuffling, called meiotic or homologous recombination, occurs thanks to DNA crossovers (COs). These crossovers are made possible by the formation of double-strand breaks (DSBs) in the DNA and their subsequent repair through a process that pairs homologous chromosomes and allows them to exchange corresponding segments of DNA. In addition to facilitating genetic shuffling, these DSBs can sometimes result in mutations due to errors in the DNA repair process. To preserve essential functions while minimizing harmful effects, DSB formation is carefully regulated in terms of timing, frequency, and location, in coordination with the structure of meiotic chromosomes. An array of proteins and protein complexes contributes to meiotic recombination and the control of DSB formation. The central meiotic DSB protein in all eukaryotes is the catalytic subunit SPO11, being directly responsible for the formation of DSBs. The aim of this master’s thesis is therefore to determine the impact of active site mutations on SPO11 DNA cleavage as well as DNA-binding activities. Previous work on the structure and functions of the yeast and mouse versions of this protein served as a base for this project. Our three main objectives are outlined as follows: first, to investigate how mutations in putative DNA-binding residues influence mouse SPO11 (mSPO11) DNA-binding activity; second, to assess the impact of active site mutations on mSPO11's cleavage activity; and third, to reconstitute double-strand break formation activity with human SPO11 (hSPO11). An additional objective is to investigate the possibility of mSPO11 and hSPO11 expression in E. coli. The first part of this project was dedicated to the Establishment of a library of mutant proteins for mSPO11 and hSPO11. The mutant selection was done based on previously identified active-site residues as well as analyses of Alphafold3-produced 3D models. The constructed expression vectors for our mutant proteins were used to create baculoviruses designed to infect SF9 insect cells and produce our mutant proteins. We successfully purified several of them, with the remaining samples available for purification. The second part of this project was dedicated to the study of the DNA-binding and DSB formation activity of the mutants. This was achieved by comparing the mutant proteins to purified wild-type SPO11. Protein-DNA interactions were analyzed using Electrophoresis Mobility Shift Assay (EMSA), while DSB formation activity was evaluated through an agarose gel DNA cleavage assay. The results obtained showed that mSPO11 possesses a sole catalytic tyrosine responsible for cutting of DNA. These results also allowed us to make progress in our understanding of the roles of some of the other residues involved in the catalytic site responsible for DSB formation. We successfully created expression vectors for mSPO11 as well as hSPO11 in E. coli. Protein expression was induced in bacteria and the resulting products were analyzed by western blot. Our results show that the expression of our mutant proteins can be effectively induced without any leaky expression before induction, and that the produced proteins are soluble. Tests to assess whether the proteins can be purified with satisfactory yields and whether their activity is comparable to that of their insect cells-produced counterparts are currently ongoing.