Abstract
Recently developed techniques allow rapid and accurate determination of protein-induced DNA unwinding. We have used such techniques to determine whether SV40 T antigen unwinds DNA. Assays were performed with purified wild type T antigen, D2 protein and the SV80 T protein over a wide range of protein concentrations. Additionally, several DNA templates were used, including SV40 DNA, pBR322 DNA, and two plasmid DNAs containing one and three copies of the SV40 origin of replication. None of the purified proteins showed unwinding activity either in the presence or absence of ATP and Mg2+. As a positive control, we found that unwinding of pBR322 DNA by Escherichia coli RNA polymerase occurred under identical assay conditions. Protein-DNA interactions that involve unwinding of the helix are sensitive to the superhelical density of the DNA. Using a filter binding assay, we found that the efficiency of the binding of T antigen to the viral origin of replication does not depend upon the topological state of the DNA. This result further supports our conclusion that SV40 T antigen is not an unwinding protein.
Highlights
It was important to determine whether T antigen bound to theSV40 origin of replication under the condicent of the DNA molecules retained on the filter by the protein
DNA mixed with wild type T antigen (O),D2 protein (0)an.d SV80 T protein (0).pBR322 DNA mixed with wild type T antigen (X)
1 ) afterone round and two rounds of wheat germ topoisomerase treatment.One microgram of covalently closed, relaxed SV40 DNA or pRR322 DNA was mixed with various concentrations of three different purified T antigens of E . coli RNA polymerase and treated with 10 units of topoisomerase
Summary
Using a filter binding assay, we found that the includes wheat germ topoisomerase (Dynan etal., 1981), efficiency of the bindingof T antigen to the viral origin catalyzes the unwinding of covalently closed circular DNA. Proteininduced unwinding of DNA can be measured by binding the protein to relaxed, covalently closed DNA and treating with another round of topoisomerase.
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