A cellular system to study responses to a collision between the transcription complex and a protein‐bound nick in the DNA template

TL;DR

This paper introduces a system to study how cells respond when RNA polymerase collides with a DNA nick mimicking a topoisomerase I complex, revealing how polymerase is ubiquitinated and degraded.

Contribution

The novel Flp-nick system allows precise in vivo study of RNA polymerase II responses to a DNA nick mimicking topoisomerase I cleavage complexes.

Findings

RNA polymerase II accumulates at the nick site when proteasome inhibition occurs with MG132.

Polymerase ubiquitination and degradation are induced by the Top1-mimicking nick complex.

The system enables detailed analysis of RNA polymerase II stalling and degradation after collision with DNA damage.

Abstract

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.

We present the cellular transcription‐coupled Flp‐nick system allowing the introduction of a Top1‐mimicking cleavage complex (Flpcc) at a Flp recognition target site within a controllable LacZ gene. LacZ transcription leads to the collision of RNA polymerase II (RNAPII) with Flpcc, and this causes RNAPII stalling, ubiquitination, and degradation. This system enables detailed studies of RNAPII destiny after collision and repair processes, with or without transcription.

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