Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2009:582:49-57.
doi: 10.1007/978-1-60761-340-4_5.

Assays for the preferential binding of human topoisomerase I to supercoiled DNA

Zheng Yang  1 , James J Champoux
Affiliations

Assays for the preferential binding of human topoisomerase I to supercoiled DNA

Zheng Yang et al. Methods Mol Biol. 2009.

Abstract

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 (3)H-labeled nicked DNA to the topoisomerase where the enzyme-DNA complexes are quantitated by the retention of the labeled DNA on a nitrocellulose membrane.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1
Gel shift assay with topo70 Y/F and topo31. Equal amounts of supercoiled circles (SC), linears (L) and nicked circles (NC) of pKSII+ plasmid DNA (0.08 pmol each) were incubated with increasing amounts of the indicated proteins in 20 μl of DNA binding buffer and analyzed by agarose gel electrophoresis as described in the text. Lanes 1, 7 and 13 contained no protein to mark the mobilities of the unbound DNAs. Lanes 2-6 contained 0.88, 1.75, 3.5, 7 and 15 pmol of topo70 Y/F protein, respectively. Lanes 8-12 contained 1.75, 3.5, 7, 15, and 30 pmol of topo31, respectively.
Fig. 2
Fig. 2
Filter binding assay. 3H-labeled nicked SV40 DNA (0.2 pmol) was incubated with topo70 Y/F and then challenged with increasing amounts of unlabeled nicked competitor (filled diamonds) or unlabeled supercoiled competitor (open diamonds) as described in the text. The percentage of 3H-labeled nicked SV40 DNA retained on the filter is plotted against the ratio of the unlabeled competitor to labeled DNA.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Champoux JJ. DNA topoisomerases: structure, function, and mechanism. Annu. Rev. Biochem. 2001;70:369–413. - PubMed
    1. Koster DA, Croquette V, Dekker C, Shuman S, Dekker NH. Friction and torque govern the relaxation of DNA supercoils by eukaryotic topoisomerase IB. Nature. 2005;434:671–674. - PubMed
    1. Muller MT. Quantitation of eukaryotic topoisomerase I reactivity with DNA. Preferential cleavage of supercoiled DNA. Biochim. Biophys. Acta. 1985;824:263–267. - PubMed
    1. Zechiedrich EL, Osheroff N. Eukaryotic topoisomerases recognize nucleic acid topology by preferentially interacting with DNA crossovers. EMBO J. 1990;9:4555–4562. - PMC - PubMed
    1. Madden KR, Stewart L, Champoux JJ. Preferential binding of human topoisomerase I to superhelical DNA. EMBO J. 1995;14:5399–5409. - PMC - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources