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To assay the preferential binding of eukaryotic type IB topoisomerases to supercoiled DNA, two methods are described that make use of a catalytically inactive mutant form of the enzyme. In the gel shift assay, the preference for binding to supercoiled plasmid DNA is detected in the presence of linear and nicked forms of the same DNA by a reduction in the mobility of the supercoiled plasmid during electrophoresis in agarose. The more quantitative filter binding assay compares the ability of nicked and supercoiled forms of the circular DNA to compete for the binding of a 3H-labeled nicked DNA to the topoisomerase where the enzyme-DNA complexes are quantitated by the retention of the labeled DNA on a nitrocellulose membrane.
Type I DNA topoisomerases release the torsional strain that builds up in DNA during essential biological processes such as DNA replication and transcription by introducing a transient single-strand break into the DNA (for review, see reference (1)). During the lifetime of the break, the enzyme is covalently attached to one end of the DNA through a phosphodiester bond to a tyrosine side chain. The type I topoisomerases are further classified into two subfamilies based on the polarity of the covalent linkage. For the type IA subfamily, the linkage is to the 5′ end of the DNA at the site of the break; this subfamily includes the prototypical bacterial topoisomerase I. The eukaryotic topoisomerases I belong to the type IB subfamily since the linkage in this case is to the 3′ end of the DNA. In the nicked state for the type IB enzymes, supercoils are relaxed by rotation of the free 5′ end of the broken strand around the intact strand of the DNA (2). The eukaryotic topoisomerases I are able to relax both negative and positive supercoils.
Human topoisomerase I has been shown to preferentially bind supercoiled DNA over relaxed DNA (3-5), providing a likely explanation for how the enzyme is targeted to torsionally-strained regions of the chromosomal DNA in the cell. An early method for detecting this preferential binding relied on measuring the amount of nicked topoisomerase I-DNA covalent complex formed after incubation of supercoiled or relaxed plasmid DNAs with the topoisomerase (3), but this method is complicated by the relaxation of the supercoiled DNA during the course of the analysis. A second method that suffers from the same weakness and is also laborious involves electron microscopic visualization of enzyme bound to DNA nodes in supercoiled DNA (4). Here we describe in detail two methods for measuring the preferential binding of topoisomerase I to supercoiled DNAs that circumvent the problem of DNA relaxation during the assay by utilizing a mutant form of the enzyme in which the active site tyrosine in the human topoisomerase I has been replaced with phenylalanine (Y723F mutation herein referred to as Y/F). In the gel shift assay, topoisomerase I binding to the DNAs in an equimolar mixture of supercoiled circles, nicked circles, and linear molecules with increasing protein concentration is detected by a reduction in the mobility of the DNA during electrophoresis in agarose. A similar assay has been described for the preferential binding of the p53 protein and HMG proteins to supercoiled DNA (6-10). The second more quantitative filter binding assay measures DNA binding by capturing labeled DNA-topoisomerase I complexes on a nitrocellulose filter (11). The relative ability of unlabeled competitor DNAs (supercoiled versus nicked) to reduce the binding of a labeled nicked DNA to the enzyme as measured by retention of the radioactive label on the filter provides a direct measure of the preference for supercoiled DNA.
An N-terminally-truncated (topo70, missing amino acids 1-174) and catalytically inactive Y/F mutant form of human topoisomerase I designated as topo70 Y/F was purified from recombinant baculovirus-infected insect SF9 cells as described (12) (see Note 1). Topo31 (containing residues 175 to 433 of human topoisomerase I) was purified as described elsewhere (13).
EcoR I (20μ/μl) (cat. R0101S) and BamH I (20μ/μl) (cat. R0136S) were purchased from New England Biolabs.
The pKSII+ plasmid DNA (Stratagene) (3.0 kb, see Note 2) was purified from plasmid-bearing Escherichia coli using the Qiagen plasmid purification kit (Qiagen Corp.). The purified DNA is free of contaminating protein and is primarily composed of negatively supercoiled DNA molecules with nicked circles representing no more than 20% of the total DNA.
Unlabeled and 3H-labeled (2-4 × 104 cpm/μg) supercoiled SV40 DNAs were isolated from SV40 infected-CV-1 cells using the Hirt procedure (14) and purified by CsCl-ethidium bromide equilibrium centrifugation (5) (see Note 3).
13 mm nitrocellulose filters with a 0.45 μm pore size (mixed cellulose ester, A045A013A) can be purchased from Advantec MFS Inc. (Dublin, CA). Set up a suction filter apparatus equipped for a 13 mm filter and adjust the vacuum for a filtration rate of 3-4 ml/min (see Note 4).
The gel shift assay for DNA binding by topoisomerase I depends on the observation that bound protein will reduce the mobility of a DNA during gel electrophoresis. Under the conditions described below, when the catalytically inactive form of human topoisomerase I (topo70 Y/F) is combined with a mixture of supercoiled, linear, and nicked circular DNAs, any selectivity of the protein for one or more of the topological forms of the DNA will result in a preferential reduction in the mobility of that DNA in the gel. This gel shift procedure provides the basis for a semiquantitative assay to detect the preferential binding of human topoisomerase I to supercoiled DNA.
In the filter binding assay, 3H-labeled nicked DNA is first incubated with the catalytically inactive form of human topoisomerase I (topo70 Y/F) (or mutant forms of the protein), followed by the addition of an unlabeled competitor DNA (either nicked or supercoiled). After an additional incubation, the fraction of labeled DNA containing bound protein is assessed by collecting the complexes on a nitrocellulose membrane. By varying the molar ratios of the unlabeled competitors to the labeled DNA, competition profiles are generated that permit one to compare the binding properties of the supercoiled and nicked DNAs for the protein of interest.
This work was supported by National Institutes of Health Grant GM49156.