Identifying novel DNA repair mechanisms during meiosis

DNA double-strand breaks (DSBs), where both strands of the DNA helix are broken at the same locus, are the most dangerous lesion to occur to the genome. DSBs must be repaired correctly and in a timely manner to ensure genomic stability. Two main mechanisms for DSB repair exist: non-homologous end-joining, whereby the ends of the DSB are cleaned up and re-ligated together, and homologous recombination, whereby a homologous template is used to repair across the generated lesion.
In the process of meiosis (the formation of gametes), programmed DSBs are introduced throughout the genome with almost all repair taking place via homologous recombination. One outcome of meiotic DSB repair is the formation of crossovers - physical linkages between homologous chromosome pairs - which enables accurate chromosome segregation at the first division. Meiotic DSB formation is catalysed by the topoisomerase like protein Spo11, which in the process of making the DSB becomes covalently attached to the DNA end. In the first stage of repair, Spo11 is removed by cleavage of the DNA upstream of where it is attached to, thereby releasing Spo11 and generating a short region of single-stranded DNA (which is further extended) making the end incompatible for repair by non-homologous end joining.
The process by which Spo11 removal takes place appears to be conserved across several species, and the enrichment of the Spo11-DNA molecules (Spo11-oligonucleotides) has enabled readouts of DSB levels and identification of regions where meiotic DSB formation has occurred. However, recent publications, along with our own unpublished data, has revealed that DSB repair takes place in mutants defective in Spo11 processing, supporting an idea that either an alternative Spo11 processing mechanisms exists, or that meiotic DSBs can form in a Spo11-idependent manner. Either of these outcomes impacts upon our current understanding of how meiotic recombination works.
The focus of this PhD project will be to identify potential candidates responsible for non-canonical meiotic recombination. Continuing work undertaken in the rotation project and from additional discussion and literature research, mutant candidates will be generated in DSB reporter strains using the yeast deletion collection, or via novel CRISPRi mechanisms or degron controlled inhibition. Following identification of potential candidates, several reporter strains will be used to identify potential repair pathways. Protein:protein interaction studies will be undertaken to identify accessory proteins involved in the repair process. Utilising expertise and knowledge from the laboratory of Dr. Ed Bolt (second supervisor) in vitro assays of purified candidate proteins on DNA substrates and enriched meiotic DNA substrates from repair mutants will be used to further define the mechanism for DSB repair.