Investigating the effect of TDP2 absence in CRISPR/Cas9 knock-out or patient-derived cell lines

The DNA repair enzyme 5’-Tyrosyl DNA phosphodiesterase 2 (TDP2) removes topoisomerase 2 peptide fromthe5’-termini of abortive cleavage complexes. TDP2 activity has been implicated in viral infection, transcriptional regulation, resistance to chemotherapy, and TDP2is mutated in Spinocerebellar Ataxia Autosomal Recessive 23 (SCAR23) syndrome. This thesis provides further evidence forthe role of TDP2 inthe DNA damage response and a better understanding of the molecular phenotypes observed in SCAR23 cells. By employing TDP2-/-cell lines, generated herein using CRISPR-Cas9, Idemonstrate the importance of TDP2 forthe repair of topoisomerase 2 induced DNA damage. Furthermore, Iidentifyand characterise two additional isoforms of TDP2, namely TDPband TDPg. TDP2bwas found to localise to the cytoplasm andnot to be involved in nuclear DNA damage repair. On the other hand, TDP2gwas found to be a pan-cellular protein and to mediate survival and DSB repair following treatment with the topoisomerase 2 ‘poison’, etoposide. In addition, Iestablish a new tyrosyl DNA phosphodiesterase(TDP)assay usingwhole cell extracts, tocompare TDP activity in different cell types and cell lines and to support ongoingdrug discoveryefforts by the Sussex Drug Discovery Group. Finally, Iidentify new human mutations in TDP2 thatdisrupt DSB repair and resistance to TOP2 induced DSBs and in so doing confirm that TDP2 mutation is associated with spinocerebellar ataxia autosomal recessive 23 (SCAR23).