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Cat. No.: VDT1-200 (for 200U)

Cat. No.: VDT1-1k (for 1KU)

Cat. No.: VDT1-5k (for 5KU)

Description

Vaccinia DNA Topoisomerase I, also known as Vaccinia Virus DNA Topoisomerase I, is a type I eukaryotic topoisomerase derived from the vaccinia virus. It catalyzes the cleavage and ligation of phosphodiester bonds at specific single-stranded DNA sequences within double-stranded DNA molecules. Vaccinia DNA Topoisomerase I possesses the enzymatic activity of relaxing supercoiled DNA, whereby it can rapidly cleave and ligate the DNA to generate double-stranded circular DNA molecules with reduced positive or negative supercoiling. Additionally, Vaccinia DNA Topoisomerase I can induce knotting or unknotting of double-stranded DNA, converting complementary single-stranded circular DNA into double-stranded circular DNA. Currently, Vaccinia DNA Topoisomerase I is commonly used as a novel tool enzyme for DNA recombinant connections, including DNA vector and recombinant fragment ligation, gapped repair, and adapter ligation.

Vaccinia DNA Topoisomerase I specifically recognizes the 5'-...(C/T)CCTT...-3' sequence within double-stranded DNA and cleaves the phosphodiester bond between the last T and the subsequent nucleotide sequence (5'-...(C/T)CCTT...-3'). During this process, the energy of the broken phosphodiester bond is retained by the formation of a covalent complex between the active site tyrosine residue (Tyr274) of Vaccinia DNA Topoisomerase I and the 3' phosphate group of the cleaved T. This complex can be attacked by the 5'-hydroxyl group of the single-stranded DNA cleaved by Vaccinia DNA Topoisomerase I, leading to the reformation of the phosphodiester bond and restoration of the original connectivity, without the requirement of ATP and DNA ligase. Simultaneously, Vaccinia DNA Topoisomerase I is released. This process of cleavage and ligation occurs repeatedly at multiple sites of the supercoiled double-stranded circular DNA, resulting in the relaxation of the supercoiling.

If the 5'-...(C/T)CCTT...-3' sequence recognized by Vaccinia DNA Topoisomerase I is located a few bases away from the 3' end of the double-stranded DNA, after the enzyme cleaves the DNA strand, the remaining single-stranded DNA with a 5'-hydroxyl group is very short and easily dissociates. In this case, the covalent complex formed between Vaccinia DNA Topoisomerase I and the 3' phosphate group of the last T in the 5'-...(C/T)CCTT...-3' sequence can also be attacked by other DNA or RNA molecules with a 5'-hydroxyl group, leading to the formation of recombinant DNA or DNA-RNA hybrid molecules. Thus, Vaccinia DNA Topoisomerase I can serve as a novel tool for in vitro DNA connections, including DNA vector and recombinant fragment ligation, gapped repair, and adapter ligation.

Source

Vaccinia DNA Topoisomerase I is obtained through recombinant expression and purification in Escherichia coli.

Purity

No detectable activities of DNA exonuclease, DNA endonuclease, and RNase other than Vaccinia DNA Topoisomerase I.

Application

Vaccinia DNA Topoisomerase I can be used to relax the supercoiled structure of closed circular DNA for subsequent enzymatic reactions, such as DNA cleavage, as well as for connecting DNA vectors and PCR amplification fragments, gapped repair, and adapter ligation.

Activity Definition

One unit of Vaccinia DNA Topoisomerase I converts 1 μg of supercoiled closed circular pUC18 DNA into the relaxed closed circular form within 30 minutes at 37°C.

10X Reaction Buffer

500 mM Tris-acetate, 1 M NaCl, 25 mM MgCl2, 1 mM EDTA, pH 7.5 at 25°C.

Enzyme Storage Solution

50 mM Tris-HCl (pH 7.5), 1 mM DTT, 0.1 mM EDTA, 0.1 M NaCl, 0.1% Triton X-100, and 50% (v/v) glycerol.

Inactivation or Inhibition

Heating at 80°C for 20 minutes can inactivate Vaccinia DNA Topoisomerase I.

Precautions

• When supercoiled plasmids are treated with Vaccinia DNA Topoisomerase I, it is recommended to use agarose gel electrophoresis without ethidium bromide (EtBr, also known as EB). Ethidium bromide or other suitable nucleic acid stains should only be used for gel staining after electrophoresis is complete. This is because when ethidium bromide is present in the gel, it binds between the bases of DNA and catalyzes the formation of supercoils. By staining the gel after electrophoresis, even if supercoiling is induced again, it will not change the electrophoretic mobility of the DNA fragments.
• This product is intended for scientific research purposes by professionals only and should not be used for clinical diagnosis or treatment, food or drug purposes, or stored in residential areas.
• For your safety and health, please wear lab attire and disposable gloves when handling this product.

Storage

Store at -20°C.