5 research outputs found
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