Abstract
Reverse gyrase introduces positive supercoils to circular DNA and is implicated in genome stability maintenance in thermophiles. The extremely thermophilic crenarchaeon Sulfolobus 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 S. islandicus 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 S. islandicus cells using a CRISPR-mediated mRNA interference approach (CRISPRi) in which topR1 mRNAs were targeted for degradation by endogenous III-B CRISPR-Cas systems. We found that the TopR1 level was reduced in the S. islandicus 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.
Highlights
DNA topoisomerases impose changes to the topological states of circular DNA and play important roles in DNA replication, recombination, and repair as well as in RNA transcription [1,2]
Because reverse gyrase is the only protein that is exclusively conserved in thermophilic organisms, the enzyme has been implicated in genome stability maintenance in thermophiles [4]
We found that methyl methanesulfonate (MMS) treatment induced a series of events including: (a) massive breaks on the sugar-phosphate backbone of genomic DNA, (b) gradual DNA content reduction and TopR1 degradation, and (c) DNA-less cell formation and chromatin protein degradation
Summary
DNA topoisomerases impose changes to the topological states of circular DNA and play important roles in DNA replication, recombination, and repair as well as in RNA transcription [1,2]. Thermophilic organisms have to deal with accelerated levels of spontaneous decomposition of DNA at high temperatures, including deamination of cytosine and base hydrolysis (usually depurination) [7,8,9]. These naturally occurring DNA lesions often lead to the formation of apurinic/apyrimidinic (AP) sites, which eventually yields single strand DNA breaks (SSBs) [7,8,9]. It has been shown that the Archaeoglobus fulgidus TopR binds to DNA nicks in vitro and inhibits heat-induced degradation of nicked DNA, and the DNA protection mechanism is independent of supercoiling [14] This raises an interesting question as to whether TopR proteins could protect damaged DNA from thermo-degradation in vivo. The strain exhibits a higher sensitivity to MMS and an early onset of DNA degradation during MMS treatment, indicating that TopR1 protects DNA breaks from thermo-degradation in vivo
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