Reverse gyrase and 3D genome architecture suppress hyperthermophile genome instability arising from horizontal gene transfer

Reverse gyrase (Rgy), a distinctive topoisomerase conserved in all hyperthermophiles, has the unique ability to introduce positive DNA supercoils. It has long been hypothesized that Rgy overwinds genomic DNA to prevent its detrimental denaturation at high temperature. However, its role in vivo has remained unresolved for more than four decades. In the course of investigating how Rgy affects genome organization in the archaeon Thermococcus kodakarensis, we find that Rgy suppresses heat-induced clustering of AT-rich genes, most of which bear signatures of horizontal gene transfer. This function depends on the topoisomerase active site of Rgy, underscoring a critical role of its action on DNA topology. The clustering of AT-rich genes is accompanied by their aberrant recruitment of the single-stranded DNA-binding protein RPA, indicative of extensive DNA melting. Genetic analysis further provides evidence that RPA sequesters denatured loci and mitigates genome instability in the absence of Rgy. We propose that DNA topology and higher-order genome organization constitute a multilayered mechanism that stabilizes horizontally acquired genes at high temperature.