DNA Supercoil Unwinding by Variola Virus Type IB Topoisomerase is Accelerated by Superhelical Density and Hindered by Enzyme-DNA Interactions

Maintenance of negative supercoils in genomic DNA is essential for diverse biological processes performed by enzymes and DNA binding proteins. Type IB topoisomerases play a key role in removing positive and negative supercoils that would otherwise accumulate at replication and transcription forks. These enzymes use a tyrosine nucleophile to cause a break in the phosphodiester DNA backbone and provide a swivel point to unwind supercoils while remaining covalently attached to the DNA via a reversible phosphotyrosine covalent linkage. An interesting mechanistic question is how topoisomerase binding, cleavage, and supercoil unwinding are regulated by the topological state of the DNA, thereby providing a mechanism for targeting the enzyme to highly supercoiled DNA domains in genomes. The variola virus type IB topoisomerase (vTopo) has unique high specificity for the target DNA sequence of 5’-CCCTT-3’ and was used to execute mechanistic studies about topoisomerase function that would be challenging to perform using other type IB topoisomerases. In these studies, we have designed and synthesized supercoiled DNA minicircles (MCs) containing a single vTopo target site, providing the first highly defined substrates for exploring the effects of superhelical density on DNA binding, reversible strand cleavage, and unwinding by type IB topoisomerases. We observe that DNA binding, cleavage, and religation are independent of superhelical density. In contrast, minicircles with low superhelical densities were found to relax more slowly than highly supercoiled minicircles, suggesting that the level of torque present in the supercoiled DNA dictates how well the rotating DNA end can be captured by the enzyme to reseal the DNA backbone. This was tested with a charge reversal K271E vTopo mutant where the Lys residue is known to interact with the rotating DNA strand. This mutant enzyme unwinds more supercoils per cleavage event, suggesting that enzyme interactions with the rotating DNA segment are important in determining the efficiency of supercoil unwinding. We infer that both superhelical density and transient contacts between vTopo and the rotating DNA determine the efficiency of supercoil unwinding. Such determinants are likely to play a role in regulating the steady-state superhelical density of DNA domains in the cell

Variola Type IB DNA Topoisomerase: DNA Binding and Supercoil Unwinding Using Engineered DNA Minicircles

Type IB topoisomerases unwind positive and negative DNA supercoils and play a key role in removing supercoils that would otherwise accumulate at replication and transcription forks. An interesting question is whether topoisomerase activity is regulated by the topological state of the DNA, thereby providing a mechanism for targeting the enzyme to highly supercoiled DNA domains in genomes. The type IB enzyme from variola virus (vTopo) has proven to be useful in addressing mechanistic questions about topoisomerase function because it forms a reversible 3′-phosphotyrosyl adduct with the DNA backbone at a specific target sequence (5′-CCCTT-3′) from which DNA unwinding can proceed. We have synthesized supercoiled DNA minicircles (MCs) containing a single vTopo target site that provides highly defined substrates for exploring the effects of supercoil density on DNA binding, strand cleavage and ligation, and unwinding. We observed no topological dependence for binding of vTopo to these supercoiled MC DNAs, indicating that affinity-based targeting to supercoiled DNA regions by vTopo is unlikely. Similarly, the cleavage and religation rates of the MCs were not topologically dependent, but topoisomers with low superhelical densities were found to unwind more slowly than highly supercoiled topoisomers, suggesting that reduced torque at low superhelical densities leads to an increased number of cycles of cleavage and ligation before a successful unwinding event. The K271E charge reversal mutant has an impaired interaction with the rotating DNA segment that leads to an increase in the number of supercoils that were unwound per cleavage event. This result provides evidence that interactions of the enzyme with the rotating DNA segment can restrict the number of supercoils that are unwound. We infer that both superhelical density and transient contacts between vTopo and the rotating DNA determine the efficiency of supercoil unwinding. Such determinants are likely to be important in regulating the steady-state superhelical density of DNA domains in the cell