Stabilization of Topoisomerase 2 Mutants Initiates the Formation of Duplications in DNA

Topoisomerase 2 (Top2) is an enzyme that helps maintain genome integrity by resolving topological structures that arise during cellular processes such as replication and transcription. To resolve these structures, a Top2 dimer creates a transient double-strand break (DSB) in the DNA. Each subunit forms a phosphotyrosyl bond with the 5’ ends of the break, and this DNA-protein intermediate is called a Top2 cleavage complex (Top2cc). Following the passage of an intact duplex, Top2 re-ligates the DNA and is released to restore genome integrity. Top2cc stabilization by chemotherapeutic drugs such as etoposide leads to persistent and potentially toxic DSBs. This thesis characterizes two novel top2 mutants, both of which are associated with a mutation signature characterized by de novo duplications. These duplication events are dependent on clean removal of the Top2cc from the DNA and DSB repair by nonhomologous end-joining. The first mutant (top2-FY,RG) was identified through a screen for etoposide hypersensitivity, and it generates a stabilized cleavage intermediate in vitro. The second mutant (top2-K720N) is the yeast equivalent of a somatic mutation in TOP2A identified in gastric cancers and choloangiocarcinomas that is also associated with a duplication mutation signature (ID17). Overall, the findings in this thesis are relevant for clinical use of chemotherapeutic drugs that target Top2 and have implications for genome evolution.