DNA Topoisomerase III from the Hyperthermophilic Archaeon Sulfolobus solfataricus with Specific DNA Cleavage Activity

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°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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Infertility and aneuploidy in mice lacking a type IA DNA topoisomerase III

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0437998100 ◽

2003 ◽

Vol 100 (5) ◽

pp. 2526-2531 ◽

Cited By ~ 55

Author(s):

K. Y. Kwan ◽

P. B. Moens ◽

J. C. Wang

Keyword(s):

Dna Topoisomerase ◽

Topoisomerase Iii ◽

Type Ia ◽

Dna Topoisomerase Iii

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Interaction between Yeast Sgs1 Helicase and DNA Topoisomerase III

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)61459-6 ◽

2000 ◽

Vol 275 (35) ◽

pp. 26898-26905

Author(s):

Richard J. Bennett ◽

Marie-Francoise Noirot-Gros ◽

James C. Wang

Keyword(s):

Dna Topoisomerase ◽

Topoisomerase Iii ◽

Dna Topoisomerase Iii

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Association of yeast DNA topoisomerase III and Sgs1 DNA helicase: Studies of fusion proteins

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.201387098 ◽

2001 ◽

Vol 98 (20) ◽

pp. 11108-11113 ◽

Cited By ~ 30

Author(s):

R. J. Bennett ◽

J. C. Wang

Keyword(s):

Fusion Proteins ◽

Dna Helicase ◽

Dna Topoisomerase ◽

Topoisomerase Iii ◽

Dna Topoisomerase Iii

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Molecular Beacon Enables Combination of Highly Processive and Highly Sensitive Rolling Circle Amplification Readouts for Detection of DNA-Modifying Enzymes

Nano LIFE ◽

10.1142/s1793984415410020 ◽

2015 ◽

Vol 05 (02) ◽

pp. 1541002 ◽

Cited By ~ 1

Author(s):

Emil L. Kristoffersen ◽

Maria Gonzalez ◽

Magnus Stougaard ◽

Cinzia Tesauro

Keyword(s):

Real Time ◽

Dna Cleavage ◽

Topoisomerase I ◽

Drug Response ◽

Molecular Beacon ◽

Rolling Circle Amplification ◽

Dna Topoisomerase ◽

Rolling Circle ◽

Topoisomerase Ib ◽

Highly Sensitive

Here we present an optimized readout format for detection of the circularized products from a DNA-based sensor for measurement of DNA-modifying enzymes including DNA Topoisomerase I. The basic design of the DNA-sensor relies on the use of a substrate that can be circularized by the activity of DNA-modifying enzymes like type IB Topoisomerases and subsequently amplified by a rolling circle amplification (RCA) mechanism. The RCA process can be followed in real-time by the addition of a molecular beacon with a fluorophore/quencher pair. Upon hybridization to the amplified product, the fluorophore/quencher pair is separated, giving rise to a fluorescent signal, measurable in pseudo real-time using a qPCR machine or in a fluorimeter. The RCA products in complex with the molecular beacon can subsequently be moved to microscopic slides and analyzed in a fluorescence microscope. We describe the proof of the principle of this molecular beacon-based method combining the qPCR readout format with the standard Rolling circle Enhanced Enzymatic Assay previously reported. Although the qPCR setup is less sensitive, it allows easy, fast, and high-throughput measurement of enzyme activities. Human Topoisomerase IB (TopIB) is a well-known target for the clinically used anticancer drugs of the camptothecin family. The cytotoxic effect of camptothecins correlates directly with the intracellular TopIB activity affecting reversibly the Topoisomerase/DNA cleavage complexes. Therefore, we envisioned that the presented method may find use for the prediction of cellular drug response and for drug screening to discover novel molecules that specifically inhibit TopIB or other DNA-modifying enzymes.

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DNA Topoisomerase I Inhibitory Activity of Quinochalcone C-glycosides from the Florets of Carthamus tinctorius

Natural Product Communications ◽

10.1177/1934578x1701201124 ◽

2017 ◽

Vol 12 (11) ◽

pp. 1934578X1701201

Author(s):

Shi-Jun Yue ◽

Wen-Xiao Wang ◽

Cheng Qu ◽

Lan-Ting Xin ◽

Yu-Ping Tang ◽

...

Keyword(s):

Inhibitory Activity ◽

Dna Cleavage ◽

Topoisomerase I ◽

Docking Study ◽

Carthamus Tinctorius ◽

In Vitro Cytotoxicity ◽

Molecular Docking Study ◽

Dna Topoisomerase ◽

Cleavage Complex

The DNA topoisomerase (Topo) I inhibitory activity of six quinochalcone C-glycosides (QCGs) isolated from the florets of Carthamus tinctorius were evaluated in vitro. Among them, anhydrosafflor yellow B (AHSYB, 4) and carthorquinoside B (6) could inhibit DNA Topo I at concentrations as low as 100 μM. Molecular docking study revealed that both of them have the capacity to stabilize Topo I-DNA cleavage complex in silico interacting with the essential binding sites, such as Arg364, Thr718 and TGP11. Besides, both compounds 4 and 6 exhibited no antitumor activity by in vitro cytotoxicity assays.

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