Reverse Gyrase Functions in Genome Integrity Maintenance by Protecting DNA Breaks In Vivo.

Reverse gyrase introduces positive supercoils to circular DNA and is implicated in genome stability maintenance in thermophiles. The extremely thermophilic crenarchaeon encodes two reverse gyrase proteins, TopR1 (topoisomerase reverse gyrase 1) and TopR2, whose functions in thermophilic life remain to be demonstrated. Here, we investigated the roles of TopR1 in genome stability maintenance in in response to the treatment of methyl methanesulfonate (MMS), a DNA alkylation agent. Lethal MMS treatment induced two successive events: massive chromosomal DNA backbone breakage and subsequent DNA degradation. The former occurred immediately after drug treatment, leading to chromosomal DNA degradation that concurred with TopR1 degradation, followed by chromatin protein degradation and DNA-less cell formation. To gain a further insight into TopR1 function, the expression of the enzyme was reduced in cells using a CRISPR-mediated mRNA interference approach (CRISPRi) in which mRNAs were targeted for degradation by endogenous III-B CRISPR-Cas systems. We found that the TopR1 level was reduced in the CRISPRi cells and that the cells underwent accelerated genomic DNA degradation during MMS treatment, accompanied by a higher rate of cell death. Taken together, these results indicate that TopR1 probably facilitates genome integrity maintenance by protecting DNA breaks from thermo-degradation in vivo.