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Development of a Novel DNA Break Capture Method to Study the Repair Dynamics of Topoisomerase-DNA Lesions Open Access

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The last decades have seen major advances in our understanding of how DNA breaks are detected, signaled to cell-cycle checkpoints, and repaired. However, a more complete understanding of how the genomic landscape influences DNA double strand break (DSB) generation and repair is needed. To address these challenges, we have developed a new method, END-seq, to measure DSBs and DNA end-resection genome-wide at base-pair resolution in vivo. By providing a snapshot of DSBs genome-wide at a given time in a population of cells, END-seq can be utilized to study the causes and consequences of genomic instability, as well as the influence of chromatin on DNA damage response. We utilized END-seq to first determine the frequency and spectrum of restriction-enzyme, zinc-finger-nuclease, and RAG-induced DSBs, and then to study the repair dynamics of topoisomerase-induced lesions. Topoisomerase II (TOP2) relieves topological stress in DNA that arises during physiological processes by introducing double strand breaks (DSBs) via a transient intermediate that contains a covalently linked DNA-bound TOP2 molecule (TOP2cc). This structural state is normally rapidly reversible, but can be stabilized by TOP2 poisons, such as the chemotherapeutic agent Etoposide (ETO), which traps TOP2ccs on DNA. Cells are thought to remove trapped TOP2ccs primarily by proteolytic degradation followed by DNA DSB repair. However, ETO is a highly reversible drug which does not permanently inhibit the enzymatic function of TOP2. Therefore, we wanted to explore if inhibiting the proteasome created conditions where TOP2cc reversibility is favored over DSB repair upon ETO washout, and what impact suppressing proteasome mediated repair of TOP2 DNA-protein adducts would have on genome stability. Here, we show that proteasome inhibitors when used in conjunction with ETO can both potentiate and suppress the genotoxic effects of ETO, depending on what order the drugs are added to the cells. Pre-treating cells with proteasome inhibitors prevented the proteolytic processing of trapped TOP2ccs, which completely protected cells from ETO-induced genome instability and preserved cellular viability, whereas treating cells with proteasome inhibitors after ETO did not. Furthermore, by preventing RNF4-mediated ubiquitination of TOP2ccs, proteasome recruitment was suppressed, which significantly enhanced genome integrity. Despite blocking DNA damage response (DDR) signaling to ETO-induced lesions, proteasome inhibitors did not prevent the formation of TOP2-linked DSBs. Rather, by suppressing the DDR to these DNA-protein adducts, TOP2 had time to uncouple itself from the DNA following ETO washout in an error-free manner that generated fewer genotoxic lesions compared to the canonical proteasome mediated repair response. Thus, inhibiting proteasome-based repair of TOP2-DNA adducts presents a possible mechanism of resistance to topoisomerase poisons.

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