Variola Type IB DNA Topoisomerase: DNA Binding andSupercoil Unwinding Using Engineered DNA Minicircles

摘要

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 vTopois unlikely. Similarly, the cleavage and religation rates of the MCswere not topologically dependent, but topoisomers with low superhelicaldensities were found to unwind more slowly than highly supercoiledtopoisomers, suggesting that reduced torque at low superhelical densitiesleads to an increased number of cycles of cleavage and ligation beforea successful unwinding event. The K271E charge reversal mutant hasan impaired interaction with the rotating DNA segment that leads toan increase in the number of supercoils that were unwound per cleavageevent. This result provides evidence that interactions of the enzymewith the rotating DNA segment can restrict the number of supercoilsthat are unwound. We infer that both superhelical density and transientcontacts between vTopo and the rotating DNA determine the efficiencyof supercoil unwinding. Such determinants are likely to be importantin regulating the steady-state superhelical density of DNA domainsin the cell.