Functional interaction of reverse gyrase with single-strand binding protein of the archaeon Sulfolobus

Abstract

Reverse gyrase is a unique hyperthermophile-specific DNA topoisomerase that induces positive supercoiling. It is a modular enzyme composed of a topoisomerase IA and a helicase domain, which cooperate in the ATP-dependent positive supercoiling reaction. Although its physiological function has not been determined, it can be hypothesized that, like the topoisomerase-helicase complexes found in every organism, reverse gyrase might participate in different DNA transactions mediated by multiprotein complexes. Here, we show that reverse gyrase activity is stimulated by the single-strand binding protein (SSB) from the archaeon Sulfolobus solfataricus. Using a combination of in vitro assays we analysed each step of the complex reverse gyrase reaction. SSB stimulates all the steps of the reaction: binding to DNA, DNA cleavage, strand passage and ligation. By co-immunoprecipitation of cell extracts we show that reverse gyrase and SSB assemble a complex in the presence of DNA, but do not make stable protein-protein interactions. In addition, SSB stimulates reverse gyrase positive supercoiling activity on DNA templates associated with the chromatin protein Sul7d. Furthermore, SSB enhances binding and cleavage of UV-irradiated substrates by reverse gyrase. The results shown here suggest that these functional interactions may have biological relevance and that the interplay of different DNA binding proteins might modulate reverse gyrase activity in DNA metabolic pathways.

Figure 1

(A) Schematic representation of 2D gel electrophoresis of a negatively supercoiled plasmid (panel 1), of negative topoisomers (panel 2) and positive and negative topoisomers (panel 3). −SC, negatively supercoiled plasmid. (B) Positive supercoiling assay: 200 ng (5 nM) of pGEM3 plasmid DNA (ΔLk > −12) were incubated with reverse gyrase purified from S.shibatae (9) for 10 min at 70°C in a final volume of 20 μl, and subjected to 2D agarose gel electrophoresis. The amounts of reverse gyrase used were: panels 1–6, 0.012, 0.024, 0.1, 0.2, 0.4 and 1.6 ng, respectively (4, 8, 34, 67, 135 and 540 pM). (C) SSB has no effect on plasmid supercoiling. Assays were performed as in (B) but reverse gyrase was omitted. Panel 1, plasmid alone; panel 2, plasmid incubated with 3 μg of SSB. (D) Effect of SSB on reverse gyrase positive supercoiling activity. Positive supercoiling assays were performed as in (B). Each reaction contained 0.1 ng of reverse gyrase (34 pM) and the following amounts of SSB: panel 1, no SSB; panels 2–6, 0.1, 0.25, 0.5, 2 and 3 μg of SSB (0.3, 0.7, 1.4, 5.5 and 8.25 μM). (E) Positive supercoiling assay as in (B), but each reaction contained 0.4 ng of reverse gyrase (135 pM) and the following amounts of SSB: panel 1, no SSB; panels 2–4, 0.1, 0.5 and 2 μg of SSB (0.3, 1.4 and 5.5 μM); panel 5, plasmid alone. P, Plasmid.

Figure 2

(A) Quantification of reverse gyrase activity; the mean specific linking difference (mean σ) was calculated as described in Materials and Methods. Reactions were performed with the indicated reverse gyrase (pM) and SSB (μM) concentrations. Values are the mean of three independent experiments. (B) Quantitation of the topoisomer distribution obtained in Figure 1E. The amount of DNA of each topoisomer is expressed as a fraction of the total amount of DNA in each reaction.

Figure 3

(A) Effect of SSB on reverse gyrase binding activity. The 3′ end-labelled RGA oligonucleotide (2.4 nM, 4 × 10⁴ c.p.m./lane) was incubated for 10 min at 37°C with purified reverse gyrase (1 ng, 350 pM) in lanes 1–5 and the following amounts of purified SSB: lane 1, no SSB; lane 2–5, 30, 60, 125 and 250 ng (0.2, 0.3, 0.7 and 1.4 μM); lanes 6–8, 60, 125 and 250 ng of SSB (0.3, 0.7 and 1.4 μM). R, reverse gyrase–DNA complexes; S, SSB–DNA complexes; P, free probe. (B) Principle of the CCA (8). (C) Effect of SSB on reverse gyrase cleavage complexes formation. The 3′ end-labelled RGA oligonucleotide was incubated and assayed by CCA with: lane 1, no protein; lane 2, purified reverse gyrase (1 ng, 350 pM); lane 3, as lane 2 but with 500 ng of SSB (1.4 μM). RG, reverse gyrase. The arrow indicates the oligonucleotide–protein covalent complex.

Figure 4

(A) Helicase assay. The 3-tailed oligonucleotide annealed to M13 DNA was incubated for 10 min at 70°C with: lane 1, no protein; lane 2, 1.6 ng of reverse gyrase (540 pM); lane 3, as lane 2 but with 250 ng of SSB (1.4 μM, corresponding to 0.5 protein molecules/binding site); lane 4, 250 ng of SSB. In lane 5 the substrate was partially denatured. Arrows indicate the annealed product and the displaced oligonucleotide. (B) Effect of SSB on reverse gyrase relaxation activity. Assays were performed as in Figure 1 but omitting ATP. Reverse gyrase 0.4 ng (135 pM) and the following amounts of SSB were used: panel 1, no SSB; panels 2–4, 100 ng, 500 ng and 2 μg (0.3, 1.4 and 5.5 μM), panel 5, plasmid alone.

Figure 5

Effect of DNA binding proteins on reverse gyrase activity. (A) Positive supercoiling assays were performed as in Figure 1, but with 0.4 ng of reverse gyrase (135 pM) and the indicated amounts of SSB and Sul7d. Considering a binding site of 6 bp for Sul7d, 2.15 μM is a saturating concentration. Thus, at the highest concentrations used the ratio SSB:Sul7d:DNA is 1:1:1. (B) Quantification of reverse gyrase activity shown in (A); numbers represent the difference of the mean σ obtained in each reaction with respect to that obtained with reverse gyrase alone (which was assigned a value 0). (C) Assays were as in Figure 1, but each panel contained 0.4 ng of reverse gyrase (135 pM), 1 μg of SSB and 0.3 μg of Sul7d; in panel 1 all proteins were added together; in panel 2 DNA was preincubated with SSB for 10 min at 70°C, then reverse gyrase and Sul7d were added; in panel 3 DNA was preincubated with Sul7d, then reverse gyrase and SSB were added. (D) Effect of Smj12 on reverse gyrase activity. Assays were as in Figure 1, but 0.4 ng of reverse gyrase (135 nM) was incubated with: panel 1, no other protein; panel 2, 0.3 μg of Smj12 (1.25 μM). (E) Quantitation of reverse gyrase activity shown in (A) and (B); the mean specific linking difference (mean σ) was calculated as described in Materials and Methods.

Figure 6

Co-immunoprecipitation of SSB and reverse gyrase. (A) Polyclonal antibody against SSB (23) was used to immunoprecipitate S.solfataricus soluble extracts. Blots were probed with polyclonal antibodies against the indicated proteins. Soluble extracts were immunoprecipitated without (lane 1) or with 50 μg/ml ethidium bromide (lane 2). Lane 3 contains 200 μg of soluble extracts (B) Polyclonal antibody against Smj12 (27) was used to immunoprecipitate S.solfataricus soluble extracts. Blots were probed with polyclonal antibodies against the indicated S.solfataricus proteins. Lane 1, immunoprecipitation and lane 2, soluble extracts (200 μg).

Figure 7

(A) Positive supercoiling activity of reverse gyrase in UV-damaged substrates in the presence of SSB. Reactions were performed as in Figure 1D, but the DNA substrate was previously irradiated with UV light (254 nm, 800 J/m²). Each reaction contained 0.1 ng of reverse gyrase (34 pM) and the following amounts of SSB: panel 1, no SSB; panels 2–5, 0.1, 0.5, 2 and 3 μg of SSB (0.3, 1.4, 5.5 and 8.25 μM); panel 6, plasmid alone. The experiment was performed in parallel to that in Figure 1D, which can be used for comparison. (B) Effect of SSB on reverse gyrase binding activity of UV-damaged DNA substrates. The 3′ end-labelled RGA oligonucleotide (4 × 10⁴ c.p.m./lane) was incubated for 10 min at 37°C with purified reverse gyrase (lanes 1–3; 1 ng, 350 pM) and the following amounts of purified SSB: lane 1, no SSB; lane 2, 125 ng of SSB (0.7 μM); lane 3, 250 ng of SSB (1.4 μM); lane 4, 125 ng of SSB (0.7 μM); lane 5, 250 ng of SSB (1.4 μM). The experiment was performed in parallel to that in Figure 3A, which can be used for comparison.