Localised activity of reverse gyrase at gene regulatory elements

ABSTRACT DNA topoisomerases are essential enzymes found in all cells, where they regulate DNA supercoiling. Reverse gyrase (RG) is a unique type of topoisomerase that introduces positive supercoils into DNA and appears exclusively in hyperthermophiles where it was proposed to play a key, yet still elusive, role. Here, we investigate RG activity in the hyperthermophilic archaeon Thermococcus kodakarensis at 85°C, its optimal growth temperature, using genetics and functional genomics assays. Deletion of RG led to a loss of positive supercoiling in plasmid DNA and the reduced dynamic range of transcription, without affecting histone occupancy. To investigate the effects of RG loss on the topology of chromosomal DNA, we established a psoralen photobinding assay (TMP-seq) in T. kodakarensis under native growth conditions. TMP enrichment patterns were consistent with the twin-domain model of transcription and further revealed that promoters of expressed transcription units are, on average, underwound. TMP-seq profiles in an RG deletion strain revealed that promoters are hotspots for RG activity, consistent with RG acting on hyper-negatively supercoiled substrates. We propose that RG acts not as a global modulator of supercoiling, but as a local genome guardian, selectively stabilising vulnerable regulatory regions to ensure a delicate balance between DNA accessibility and integrity under extremely high temperatures. Competing Interest Statement The authors have declared no competing interest.