Wednesday, April 2, 2025
4/2/2025
Biochemical determinants of genome instability induced by eukaryotic topoisomerase II - Project summary DNA topoisomerases carry out changes in DNA structure needed for transcription, replication, and maintenance of DNA structure. Topoisomerases introduce transient breaks in DNA with a protein/DNA covalent intermediate termed the cleavage complex. This cleavage mechanism allows cells to carry out changes in DNA conformation such as relaxation of DNA supercoiling without the dangers of frank DNA double strand breaks. Nonetheless, topoisomerase-induced breaks can persist due to small molecules that interfere with the topoisomerase reaction, due to alternate DNA structures, or due to mutations within the topoisomerase protein. Our recent work led to the identification of mutations in eukaryotic Top2 that generate high levels of Top2 cleavage complexes in the absence of small molecule inhibitors (hcTop2). Importantly, we found that expression of hcTop2 mutants in yeast is mutagenic and causes de novo duplications of 2-5 nucleotides. This pattern of duplications is very similar to ID17, a mutational signature found in some cancer cells. Cells with the ID17 pattern carry a point mutation in human Top2, Top2K743N. We constructed the yeast ortholog yTopK720N and showed that the purified protein gives rise to elevated levels of Top2 cleavage and that expression of the mutant protein in yeast cells leads to a pattern of mutations that was similar to ID17. These hcTop2 proteins can be applied to address fundamental questions concerning how topoisomerases regulate cleavage reactions, and how they impact genome stability. A major question that we plan to address in this work is to determine how the biochemical properties of a topoisomerases dictate where genome instability occurs. We hypothesize that a combination of the biochemical properties of topoisomerases, along with other factors such as chromatin structure and DNA repair pathways are likely to determine important aspects of genome instability induced by the enzymes. We developed an assay to assess sites of DNA cleavage using reactions with hcTop2 mutant proteins in vitro followed by detection of cleavage sites by next generation sequencing. Since we have identified many mutations induced in yeast by hcTop2 expression, we can address whether hotspots of mutation induced by Top2 correlate with strong Top2 cleavage sites in vitro. Notably, not all hcTop2 mutants give rise to the same mutation spectrum, implying that the Top2 enzyme plays a direct role in the sites of induced mutations. Since we identified mutations in human Top2 andTop2 that are hyper cleavage, we can use these mutants to address whether human Top2 proteins give rise to the same pattern of mutations as seen with yeast Top2. Finally, the hcTop2 mutants can be applied as a tool to identify pathways to repair Top2-induced DNA damage. Since hcTop2 mutants in both Top2 and Top2 are present in cancer genome database, we hypothesize that these hcTop2 mutants can be drivers of genome instability and carcinogenesis in mammalian cells. These Top2 mutants may reveal new vulnerabilities of cancer cells that express mutant DNA metabolic enzymes.
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