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. 2003 Sep;185(18):5500-7.
doi: 10.1128/JB.185.18.5500-5507.2003.

DNA topoisomerase III from the hyperthermophilic archaeon Sulfolobus solfataricus with specific DNA cleavage activity

Penggao Dai  1 , Ying Wang, Risheng Ye, Liang Chen, Li Huang
Affiliations

DNA topoisomerase III from the hyperthermophilic archaeon Sulfolobus solfataricus with specific DNA cleavage activity

Penggao Dai et al. J Bacteriol. 2003 Sep.

Abstract

We report the production, purification, and characterization of a type IA DNA topoisomerase, previously designated topoisomerase I, from the hyperthermophilic archaeon Sulfolobus solfataricus. The protein was capable of relaxing negatively supercoiled DNA at 75 degrees C in the presence of Mg2+. Mutation of the putative active site Tyr318 to Phe318 led to the inactivation of the protein. The S. solfataricus enzyme cleaved oligonucleotides in a sequence-specific fashion. The cleavage occurred only in the presence of a divalent cation, preferably Mg2+. The cofactor requirement of the enzyme was partially satisfied by Cu2+, Co2+, Mn2+, Ca2+, or Ni2+. It appears that the enzyme is active with a broader spectrum of metal cofactors in DNA cleavage than in DNA relaxation (Mg2+ and Ca2+). The enzyme-catalyzed oligonucleotide cleavage required at least 7 bases upstream and 2 bases downstream of the cleavage site. Analysis of cleavage by the S. solfataricus enzyme on a set of oligonucleotides revealed a consensus cleavage sequence of the enzyme: 5'-G(A/T)CA(T)AG(T)G(A)X / XX-3'. This sequence bears more resemblance to the preferred cleavage sites of topoisomerases III than to those of topoisomerases I. Based on these data and sequence analysis, we designate the enzyme S. solfataricus topoisomerase III.

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Figures

FIG. 1.
FIG. 1.
Phylogenetic relationship of type IA DNA topoisomerases from Archaea, Bacteria, and Eucarya. Multiple-sequence alignments were obtained by using ClustalW. The tree was constructed by using the neighbor-joining method. Bootstrap values were obtained with 1,000 replicates. The source organism and accession number for each protein are as follows: Sso, S. solfataricus (Topo III: G90241; Top R: F90247); Pfu, Pyrococcus furiosus (Topo III: O73954; Top R: NP_578224); Ape, A. pernix (Topo III: Q9YB01); Mth, Methanobacterium thermautotrophicum (Topo III: O27661); Mja, M. jannaschii (Topo III: Q59046); Tma, T. maritima (Topo I: P46799; Top R: C72409); Fis, Fervidobacterium islandicum (Topo I: O34204); Eco, E. coli (Topo I: P06612; Topo III: P14294); Hin, Haemophilus influenzae (Topo I: P43012; Topo III: P43704); Pmu, Pasteurella multocida (Topo I: Q9CN30; Topo III: Q9CP53); Vch, Vibrio cholerae (Topo I: Q9KRB2; Topo III: Q9KQF5); Cel, Caenorhabditis elegans (Topo III: O61660); Sce, S. cerevisiae (Topo III: P13099); and Hsa, Homo sapiens (Topo IIIα: Q13472). Sso Topo III, Pfu Topo III, Ape Topo III, Mth Topo III, and Mja Topo III were previously known as Sso Topo I, Pfu Topo I, Ape Topo I, Mth Topo I, and Mja Topo I, respectively. The topoisomerase domain of reverse gyrase, indicated by amino acid residues in parentheses, was used in the analysis.
FIG. 2.
FIG. 2.
Purification and topoisomerase activities of Sso Topo III. (A) SDS-polyacrylamide gel electrophoresis of purified Sso Topo III. A sample (1.5 μg) of purified Sso Topo III was electrophoresed through an SDS-polyacrylamide gel (10% acrylamide). The gel was silver stained. Molecular masses of protein standards are indicated. (B) DNA relaxation by Sso Topo III. Serial dilutions of Sso topo III were incubated at 75°C for 30 min with pUC18 (300 ng) in 50 mM Tris-HCl, pH 8.8, 1 mM DTT, 0.1 mM EDTA, 10 mM MgCl2, 90 mM NaCl, 30 μg of BSA/ml, and 12% (vol/vol) ethylene glycol. Reaction mixtures were electrophoresed through 1.5% agarose. N, nicked plasmid; S, supercoiled plasmid. (C) Determination of the end point of DNA relaxation catalyzed by Sso Topo III. Sso Topo III (2 pmol) was incubated with pUC18 (300 ng) at 75°C for 30 min in the standard DNA relaxation assay (lane 1). Single-nick pUC18 (1 μg, lane 3) was incubated with Pfu DNA ligase (4 Weiss units) under the same conditions as those used for DNA relaxation to yield a completely relaxed plasmid (lane 2). Samples were electrophoresed in agarose. (D) Mutational analysis of the putative active site of Sso Topo III. A sample of wild-type or mutant Y318F enzyme was incubated with pUC18 (300 ng) at 75°C for 30 min in the standard DNA relaxation assay. Products were analyzed by agarose gel electrophoresis.
FIG. 3.
FIG. 3.
Characterization of the DNA relaxation activity of Sso Topo III. (A) Effect of temperature on DNA relaxation by Sso Topo III. Relaxation reactions were performed at indicated temperatures for 30 min. Reaction products were resolved by electrophoresis in agarose. (B) Effect of divalent cations on DNA relaxation by Sso Topo III. Reactions were performed at 75°C for 30 min in the presence of a tested divalent cation (10 mM). (C) Effect of Mg2+ concentration on DNA relaxation by Sso Topo III. Reactions were performed at 75°C for 30 min with various concentrations of MgCl2. (D) Effect of NaCl on DNA relaxation by Sso Topo III. Reactions were performed at 75°C for 30 min with various concentrations of NaCl. C, a control in which negatively supercoiled pUC18 DNA alone was electrophoresed. (E) Effect of spermidine on DNA relaxation by Sso Topo III. Reactions were carried out under the standard assay conditions at various concentrations of spermidine. C, control.
FIG. 4.
FIG. 4.
Identification of the site of oligonucleotide cleavage by Sso Topo III. (A) The sequence of the 40-nt cleavage substrate. The cleavage site is indicated by the arrow. (B) Cleavage of the 40-mer by Sso Topo III. Sso Topo III (2 pmol) was incubated with the 5′ end radiolabeled 40-mer (0.1 pmol) at 75°C for 30 min in 50 mM Tris-HCl, pH 8.8, 1 mM DTT, 0.1 mM EDTA, 10 mM MgCl2, 90 mM NaCl, 30 μg of BSA/ml, and 12% (vol/vol) ethylene glycol. Reaction mixtures were analyzed by urea-polyacrylamide gel electrophoresis. Lane 1, 40-mer; lane 2, cleavage reaction products; lane 3, oligonucleotide size markers (13-mer [AATTCAGCAAGGC] and 11-mer [AATTCAGCAAG]); and lane 4, a mixture of the cleavage products and the size markers.
FIG. 5.
FIG. 5.
Characterization of the oligonucleotide cleavage activity of Sso Topo III. (A) Effect of temperature on DNA cleavage by Sso Topo III. Cleavage reactions were performed at indicated temperatures for 30 min. Reaction products were resolved by urea-polyacrylamide gel electrophoresis. C, a control in which the oligonucleotide substrate was incubated for 30 min at 75°C. (B) Effect of divalent cations on DNA cleavage by Sso Topo III. Reactions were performed at 75°C for 30 min in the presence of a tested divalent cation (2 mM). C, control. (C) Effect of Mg2+ concentration on DNA cleavage by Sso Topo III. Reactions were performed at 75°C for 30 min at various concentrations of MgCl2. C, control. (D) Effect of salt concentration on DNA cleavage by Sso Topo III. Reactions were performed at 75°C for 30 min at various concentrations of NaCl.
FIG. 6.
FIG. 6.
Determination of the sequence requirement of the Sso Topo III-catalyzed oligonucleotide cleavage. The 40-mer was successively shortened from both ends to yield eight oligonucleotides. As illustrated at the top of the figure, each oligonucleotide is represented by a horizontal line proportional in length to the size of the oligonucleotide. The point of cleavage is shown by a vertical arrow. The number of bases 5′ or 3′ of a cleavage site is indicated by the number on the left or right, respectively, of the arrow. Each oligonucleotide was incubated at 75°C for 30 min with (+) or without (−) Sso Topo III (2 pmol). The cleavage products of the oligonucleotide containing 13 bases 5′ and 2 bases 3′ of the cleavage site were analyzed by electrophoresis in 25% polyacrylamide containing 7 M urea, whereas those from other oligonucleotides were resolved in 19% polyacrylamide gels.
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