Trapped Topoisomerase II initiates formation ofde novoduplicationsviathe nonhomologous end-joining pathway in yeast

Abstract

Topoisomerase II (Top2) is an essential enzyme that resolves catenanes between sister chromatids as well as supercoils associated with the over-or under-winding of duplex DNA. Top2 alters DNA topology by making a double-strand break (DSB) in DNA and passing an intact duplex through the break. Each component monomer of the Top2 homodimer nicks one of the DNA strands and forms a covalent phosphotyrosyl bond with the 5¢ end. Stabilization of this intermediate by chemotherapeutic drugs such as etoposide leads to persistent and potentially toxic DSBs. We describe the isolation of a yeast top2 mutant (top2-F1025Y,R1128G) whose product generates a stabilized cleavage intermediate in vitro. In yeast cells, overexpression of the top2-F1025Y,R1128G allele is associated with a novel mutation signature that is characterized by de novo duplications of DNA sequence that depend on the nonhomologous end-joining pathway of DSB repair. Top2-associated duplications are promoted by the clean removal of the enzyme from DNA ends and are suppressed when the protein is removed as part of an oligonucleotide. TOP2 cells treated with etoposide exhibit the same mutation signature, as do cells that over-express the wild-type protein. These results have implications for genome evolution and are relevant to the clinical use of chemotherapeutic drugs that target Top2.

Recurrent mutations in topoisomerase 2a cause a novel mutator phenotype in human cancers

“…This suggests that other TOP2A alterations might induce mutator phenotypes similar to ID_TOP2A [17]. Interestingly, this yeast model also showed a strong increase in sensitivity to the topoisomerase II inhibitor etoposide [17]. As we observed in a human TOP2A p.K743N tumor, the yeast double mutants showed strong increases in SVs.…”

“…Although we have only observed this pattern of indel mutagenesis in human tumors carrying the TOP2A p.K743N substitution, a TOP2 double-mutant (p.F1025Y and p.R1128G) in yeast (Saccharomyces cerevisiae) was reported to have a similar mutator phenotype. This suggests that other TOP2A alterations might induce mutator phenotypes similar to ID_TOP2A [17]. Interestingly, this yeast model also showed a strong increase in sensitivity to the topoisomerase II inhibitor etoposide [17].…”

“…Furthermore, neither of the amino acids altered in TOP2 p.F1025 or p.R1128 is conserved in human TOP2A or TOP2B. Moreover, TOP2 overexpression in yeast also resulted in indels similar to both ID_TOP2A and the indels observed in the p.F1025 and p.R1128 double mutant [17]. Given that such different alterations, as well as overexpression of wild-type TOP2, generated similar indel mutator phenotypes suggests that still other TOP2A alterations might result in similar mutagenesis.…”

“…show that ID_TOP2A almost certainly contributed to tumorigenesis. Data from a yeast model suggest that presence of ID_TOP2A might indicate increased tumor vulnerability to topoisomerase II inhibitors such as etoposide [17].…”