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. 2018 Jan 25;46(2):861-872.
doi: 10.1093/nar/gkx1247.

TopA, the Sulfolobus solfataricus topoisomerase III, is a decatenase

Anna H Bizard  1 , Xi Yang  2   3 , Hélène Débat  3   4 , Jonathan M Fogg  5   6   7 , Lynn Zechiedrich  5   6   7 , Terence R Strick  2   3   8 , Florence Garnier  3   4 , Marc Nadal  2   3
Affiliations

TopA, the Sulfolobus solfataricus topoisomerase III, is a decatenase

Anna H Bizard et al. Nucleic Acids Res. .

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.

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Figures

Figure 1.
Figure 1.
Activity of SsoTopA in bulk experiments. (A) For relaxation, the protein/pTZ18R DNA molar ratio used was 1:1 and the incubation time was as indicated. The reaction products were analyzed by one dimensional agarose gel electrophoresis. (B) Decatenation of kDNA was carried out by adding SsoTopA just before the incubation at the indicated temperature for 30 min in the presence of kDNA. The reactions were stopped and kDNA products were separated on a 2% agarose gel. OC indicates open circular DNA, Rel, the relaxed topoisomers and -SC the negatively supercoiled DNA for the pTZ18R plasmid (A) or the mCs (B).
Figure 2.
Figure 2.
Activity of SsoTopA at very high temperatures. (A) The DNA controls (lanes Ct°c) were incubated in the absence of SsoTopA for 8 min at the indicated temperatures. (B) The pTZ18R substrate was incubated at the temperature indicated in the presence of SsoTopA (at a molar ratio of 2:1). C75 corresponds to the DNA control incubated at 75°C for 8 min without SsoTopA. The reaction products were analyzed by one dimensional agarose gel electrophoresis: OC indicates open circular DNA, Rel, the relaxed topoisomers and -SC the negatively supercoiled DNA form. FI* and FI** correspond to unlinked DNA species and were apparently similar to highly unwound species first described by Parada and Marians (42).
Figure 3.
Figure 3.
Analysis of FI* and FI** DNA. (A) Generation of FI* and FI** over time during incubation with SsoTopA at 95°C. After incubation at 95°C for the indicated times, samples were cooled at 4°C for 5 min, centrifuged before adding 0.1% SDS, 25 mg/ml bromophenol blue and 15% sucrose (final concentrations). The samples were further incubated for an additional 5 min at 4°C. The samples were loaded onto an agarose gel at 4°C and run at 3 V/cm for 6 h at 4°C. (B) Spontaneous re-annealing of single-stranded forms FI** at 25°C. The samples were treated as in (A) except that they were further incubated for an additional 5 min at 25°C instead of 4°C prior to loading on the gel. (C) Sensitivity of the different DNA forms to S1 nuclease. Purified pTZ18R DNA obtained after incubation with TopA at 95°C for 4 min was further incubated at 4°C overnight without (0) or with 1 × 10−3 unit or 10 × 10−3 unit of S1 nuclease. (D) Sensitivity of the different DNA forms to T5 exonuclease. pTZ18R was linearized by BamHI (lanes 1–4) and then heated at 95°C for 5 min (lanes 2–4). Unlinked DNA forms (FI* and FI**) were obtained after incubation of pTZ18R with SsoTopA at 95°C for 5 min (lanes 6–8). After incubation at 95°C the DNA was further incubated at 4°C without (lanes 1, 2 and 6) or with 10 units of T5 exonuclease for 4 h (lanes 3 and 7) or 16 h (lanes 4 and 8). The SsoTopA treated samples (lanes 6–8) were additionally heated at 95°C for 2 min just before loading. Lane 5 is the control DNA substrate (pTZ18R). In the different experiments (A–D), the reaction products were analyzed by one dimensional gel electrophoresis: OC indicates open circular DNA, Lin the linear double stranded DNA, SSLin the linear single-stranded DNA, -SC the negatively supercoiled DNA form and the particular FI* and FI** forms.
Figure 4.
Figure 4.
Reversible activity of SsoTopA. (A) Kinetics of DNA melting catalyzed by SsoTopA. The pTZ18R substrate relaxed by SsoTopA (molar ratio of 2:1) at 75°C was further incubated at 95°C in the presence of TopA for the time indicated above each corresponding lane. (B) Kinetics of DNA re-annealing catalyzed by TopA. The reaction mix (SsoTopA and resulting products obtained after 8 min at 95°C, panel (A) was further incubated at 75°C. The incubation times are indicated above each corresponding lane. The reaction products were analyzed by one dimensional agarose gel electrophoresis: OC indicates open circular (nicked) DNA, Rel, the relaxed topoisomers and -SC the negatively supercoiled DNA. FI* and FI** are the unlinked DNA species.
Figure 5.
Figure 5.
Decatenation of multi-linked DNA circles by SsoTopA. (A) The PP used to produce catenated molecules (CP) is schematically represented. The catenated DNA is composed of the LC (in blue color) and the mC (in red color) which are interlinked. The different enzymes used to characterize the different forms of these DNA are indicated. (B) The multi-linked DNA substrate (lanes 5 and 7) produced from the parent plasmid pMC339 (PP) includes two major species, the PP itself and the catenane (CP), and one minor species, the decatenated LC and mC. The multi-linked DNA substrate was incubated for 1 h at 37°C with BamHI (lane 1), NdeI (lanes 2–4), Nt.BspQI (lane 6) or Nb.BbvCI (lanes 10–12). Sso TopA was added in lanes 3, 4, 8, 9, 11 and 12 and incubated for 20 min at 80°C (lanes 3, 8 and 11) or 90°C (lanes 4, 9 and 12) producing relaxed DNA. The vertical plain bars correspond to the large plasmid relaxed at 80°C by TopA and the vertical dashed line corresponds to a mix of large plasmid and catenane relaxed at 80°C by SsoTopA. The DNA was loaded onto a 1.7% agarose gel and ran at about 2.5 V cm−1 for 3 h. The different DNA bands were attributed according to their size and topological forms (OC, open circular; Lin, linear; Rel, relaxed; -SC, negatively supercoiled) as deduced from the control reactions. M corresponds to the GeneRuler 1 kb ladders (ThermoFisher).
Figure 6.
Figure 6.
Relaxation activity of SsoTopA on single molecule. (A) Time-trace of a DNA extension variation at 45°C of a 3 kb DNA extended by a force of 0.45 pN in the presence of 600 pM of SsoTopA. The time at which the negative supercoils are added to the DNA by rotating the magnet, is indicated by a red arrow. Extension length fluctuations are indicated by green arrows and the relaxation burst by black arrows. (B) Histograms of the average time in second per supercoil removed. The red line is the best fit (P-value = 0.966) using the single exponential equation formula image, where formula image is the average time. A total of 148 relaxation events were used in this analysis. (C) Histograms of the burst processivity of SsoTopA. The number of supercoils removed was obtained from the extension variation using, for each bead, its calibration curve (39). The periodicity of one for the supercoil removal is indicated above the peaks. A total of 148 relaxation events were used in this analysis.
Figure 7.
Figure 7.
Relaxation activity of SsoTopA on single molecule containing a permanent single-stranded DNA bubble. (A) Time-trace of the DNA extension variation at 35°C of the 2.2 kb DNA extended by a force of 0.42 pN and in the presence of 6 pM SsoTopA. (B) Histograms of the burst processivity of SsoTopA on a DNA containing a bubble. The number of supercoils removed was obtained from the extension variation using the calibration curve.
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