We present a transcription-coupled Flp-nick system enabling a stable protein-bound nick mimicking a topoisomerase I-DNA cleavage complex. The nick is introduced at a single site within a controllable LacZ gene inserted into the Saccharomyces cerevisiae genome. This system allows unique single-site studies of a frequently occurring damage within a transcription unit in vivo. As proof of principle, we demonstrate RNA polymerase II accumulation at the damage site when MG132 inhibits the proteasome. Similarly, accumulation occurs when polymerase ubiquitination is abolished by deletion of the ubiquitinase ELC1 gene. This indicates that a topoisomerase I-DNA mimicking cleavage complex per se induces RNA polymerase II ubiquitination and degradation. These findings advance understanding of cellular responses to topoisomerase I-targeting drugs used in cancer chemotherapy.

• PMID: 40309784
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Reference Type

Journal Article

Authors

Herring P, Roedgaard M, Holst CM, Christensen H, Knudsen BR, Bjergbaek L, Andersen AH

Primary Lit For

Additional Lit For

FLP1 | ELC1 | DNA-directed RNA polymerase II complex | flp1-H305L

Review For