Nucleic Acids Research, Vol 27, Issue 4 984-992, Copyright © 1999 by Oxford University Press
KL West and CA Austin
We have used gel retardation analysis to show that human DNA topoisomerase
IIbeta can bind a 40 bp linear duplex containing a single DNA topoisomerase
IIbeta cleavage site. Furthermore, we demonstrate for the first time that
human DNA topoisomerase IIbeta binds to four-way junction DNA. This
supports previous suggestions that topoisomerase II may be targeted to
supercoiled DNA through the recognition of DNA cruciforms, helix-helix
crossovers and hairpins. DNA topoisomerase IIbeta had a 4-fold higher
affinity for the four-way junction than for the linear duplex, as
demonstrated by protein titration and competition analysis. Furthermore,
the DNA topoisomerase IIbeta:four-way junction complex was significantly
more salt stable than the complex with linear DNA. The four-way junction
contained potential topoisomerase IIbeta cleavage sites straddling the
points of strand exchange, and indeed, topoisomerase IIbeta was able to
cleave three of these four predicted sites. This indicates that
topoiso-merase IIbeta can bind to the centre of the junction. Topoisomerase
II has to bind both the transported and the gated DNA helices prior to
strand passage, and it is possible that both helices are provided by the
four-way junction in this case. The stable complex of DNA topoisomerase
IIbeta with four-way junction DNA may provide an ideal substrate for
further studies into the mechanism of substrate recognition and binding by
DNA topoisomerase II.
ARTICLES
Human DNA topoisomerase IIbeta binds and cleaves four-way junction DNA in vitro
School of Biochemistry and Genetics, The Medical School, The University of Newcastle Upon Tyne, Newcastle Upon Tyne NE2 4HH, UK.
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