Product Description

Topoisomerase I (Topo I) from vaccinia virus is a purified enzyme, free of detectable exo- and endonuclease and RNase activities. It has a molecularmass of 32 kDa..Topoisomerase I relaxes supercoiled DNA molecules. The enzyme initiates transient breakages and rejoins of phosphodiester bonds in superhelical turns of closed-circular DNA. Enzyme activity is independent of right- and left-handed superhelices.

 Topoisomerase I plays a major role in critical cellular processes by catalyzing the breakage and religation of phosphodiester bonds in a single strand of DNA. This results in the removal of supercoils (either positive or negative superhelical turns).

Topo I from vaccinia virusis a type I eukaryotic topoisomerase that cleaves DNAto the target sequence [5’(C/T)CCTT↓].¹ Cleavage of the strand containing this sequence occurs by a transesterification reaction in which a covalent bond is formed between a tyrosine on the Topo I and the 3-phosphate of the last thymidine of the targetsequence. The other DNA strand is not cleaved. Subsequent religation of the phosphodiester bond results in DNA with fewer superhelical turns.

 Topo I does not require Mg²⁺ to function, but low concentrations of this cation may increase its activity. If the (C/T)CCTT site is within a few bases of the end of the DNA molecule, those bases 3’ of the nick dissociate from the Topo I-DNA complex. Topo I may then create a recombinant molecule by joining the cleaved (C/T)CCTT-containing DNA with another DNA duplex if the other DNA duplex can basepair with the noncleaved strand.^2,3 Topo I can also form recombinant molecules with the 5-end of RNA molecules.³ Topoisomerase I is important in studying vital processes including replication, transcription, and recombination.⁴ The enzyme may be used to study DNA structure and topology such as: the effects of supercoiling on transcription in vitro, chromatin reconstitution in vitro, and

the degree of supercoiling of DNA. It can also be used to assay mutant plasmids which differ in length by only one base-pair and to increase restriction endonuclease digestion of resistant DNA substrates by unwinding the DNA coils to expose restriction sites.