TopA, the Sulfolobus solfataricus topoisomerase III, is a decatenase

Abstract

DNA topoisomerases are essential enzymes involved in all the DNA processes and among them, type IA topoisomerases emerged as a key actor in the maintenance of genome stability. The hyperthermophilic archaeon, Sulfolobus solfataricus, contains three topoisomerases IA including one classical named TopA. SsoTopA is very efficient at unlinking DNA catenanes, grouping SsoTopA into the topoisomerase III family. SsoTopA is active over a wide range of temperatures and at temperatures of up to 85°C it produces highly unwound DNA. At higher temperatures, SsoTopA unlinks the two DNA strands. Thus depending on the temperature, SsoTopA is able to either prevent or favor DNA melting. While canonical topoisomerases III require a single-stranded DNA region or a nick in one of the circles to decatenate them, we show for the first time that a type I topoisomerase, SsoTopA, is able to efficiently unlink covalently closed catenanes, with no additional partners. By using single molecule experiments we demonstrate that SsoTopA requires the presence of a short single-stranded DNA region to be efficient. The unexpected decatenation property of SsoTopA probably comes from its high ability to capture this unwound region. This points out a possible role of TopA in S. solfataricus as a decatenase in Sulfolobus.

The reverse gyrase TopR1 is responsible for the homeostatic control of DNA supercoiling in the hyperthermophilic archaeon Sulfolobus solfataricus

“…These results are confirmed by the migration of the corresponding products in a bidimensional gel (Figure d, 80 and 88°C). An additional product is detected only at 88°C, the form F1* corresponding to highly unwound DNA produced by TopA only at high temperatures as previously described (Bizard et al, ).…”

“…These results are confirmed by the migration of the corresponding products in a bidimensional gel(Figure 4d, 80 and 88°C). An additional product is detected only at 88°C, the form F1* corresponding to highly unwound DNA produced by TopA only at high temperatures as previously described(Bizard et al, 2018).TopR1 is active at 80°C by generating progressively intermediate negatively and positively supercoiled forms as indicated by the double bands co-migrating at each level of topoisomers and more easily distinguishable in the vicinity of the topoisomers close to the open circular form (Figure 4b). This profile is representative of a distributive activity of TopR1 first progressively relaxing negative supercoils and further introducing positive supercoils into the DNA plasmid as previously described (Bizard et al, 2011).…”

“…We have observed that TopoVI is much less active at 88°C than 80°C for decatenating DNA, suggesting TopoVI alone might not be sufficient to assume this role in actively dividing cells at 88°C. Interestingly, we have previously reported that TopA is even more active in decatenating DNA at high temperatures (Bizard et al, 2018). Thus, TopA might punctually replace and/or assist TopoVI for this function to achieve properly the cell division with a correct segregation of sister chromosomes at 88°C.…”

“…The three type of IA topoisomerases were purified from S. solfataricus as described previously (Bizard et al, 2011(Bizard et al, , 2018 and TopoVI from a closely related strain, S. shibatae, as described previously (Buhler, Gadelle, Forterre, Wang, & Bergerat, 1998…”

“…In addition, TopR2 was reported to be a processive enzyme, while TopR1 is a distributive one (Bizard et al, ). TopA relaxes negative supercoils (Bizard et al, ; Dai, Wang, Ye, Chen, & Huang, ). TopoVI is able to relax both negative and positive supercoils as previously described in a closely related strain Sulfolobus shibatae (Bergerat, Gadelle, & Forterre, ).…”