Synthetic interaction screening in fission yeast using saturated transposon analysis

Thesis

Genomes are regularly exposed to endogenous and exogenous insults that result in DNA damage and threaten the completion of DNA replication, chromosomal stability and error-free chromosome segregation, all of which have implications in genetic disorders and cancer in humans. A major way of promoting and/or maintaining genome stability in cells involves the crucial process of homologous recombination (HR). Historically, various yeast genetic screens, from first-generation to next-generation approaches, have contributed to the identification of genes functioning in DNA repair, replication and recombination pathways. Considering HR is a versatile repair pathway, functioning in double-strand break repair, replication fork repair, and interstrand cross-link repair in mitotic cells, the genetic profiles of the molecular players of HR are not complete. Therefore, this study aimed to adopt a transposon sequencing method called SATAY (Saturated Transposon Analysis in Yeast), which combines transposon insertion mutagenesis with deep-sequencing of transposon-genome junctions in the fission yeast Schizosaccharomyces pombe and utilise it to screen for genes involved in recombination. By using the Ac/Ds transposase/transposon system from maize, SATAY was successfully developed in the wild-type background with 584,505 unique miniDs insertions mapped throughout the genome, generating a list of essential and non-essential genes for cell viability. To screen for novel factors involved in HR, SATAY was used in a series of synthetic interaction screens in mus81∆, fml1∆ and the single-strand DNA annealing-defective rad52-R45A mutant backgrounds. Interestingly, following the optimisation of SATAY as a genetic screening tool in the mus81∆ background, the sor1 gene, encoding the WAPL antagonist during cohesin-mediated sister chromatid cohesion, was identified as a putative interactor of mus81, displaying a growth defect and enhanced camptothecin sensitivity as a double mutant. In addition to investigating the molecular portfolio of HR, this study also shows an initial attempt at screening for genes associated with RTS1 replication fork barrier-induced recombination-dependent replication in the context of replication fork repair during stress using a modified SATAY protocol.

Authors: Senin, B

• DOI: 10.5287/ora-mzyam7xxj
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: synthetic interaction screening; Fission Yeast; DNA repair; recombination-dependent replication; transposon insertion sequencing (Tn-seq); homologous recombination
• Subjects: Method Development; Bioinformatics; Yeast genetics; Molecular genetics; Molecular biology